miRCURY LNA™ Universal RT
microRNA PCR
Instruction manual v6.2
#203301-203351
July 2016
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Table of contents
Product summary
I. Reagent kits
II. Primer Sets and Panels
Storage
Additional required materials
Recommended accompanying products
3
4
6
10
10
11
Product description
Control Assays
12
13
Before starting the experiment
17
Protocols
A. Individual assays
B. Human and Mouse&Rat microRNA PCR Panels
C. Focus microRNA PCR Panels
D. Pick-&-Mix microRNA PCR Panels
20
20
29
36
43
Tips to protocol
52
Troubleshooting guide
60
FAQs
61
Related products
63
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Product summary
The miRCURY LNA™ Universal RT microRNA PCR system is a microRNA-specific,
LNA™-based system designed for sensitive and accurate detection of microRNA by
quantitative real-time PCR using SYBR® Green. The method is based on universal reverse
transcription (RT) followed by real-time PCR amplification with LNA™ enhanced primers
(for more details please see page 12). The miRCURY LNA™ Universal RT microRNA PCR
portfolio is comprised of four types of reagent kits; including:
• Universal cDNA synthesis kit II
• RNA Spike-in kit
• ExiLENT SYBR® Green master mix kit
• microRNA primer sets available in pre-defined Human, Mouse&Rat and Focus PCR panels and
customized Pick-&-Mix PCR panels as well as individual primer sets and reference genes. All PCR
panels are Ready-to-Use delivered with one 10 μL PCR reaction per well.
Product overview
Universal cDNA synthesis kit II
Description page 4
RNA Spike-in kit
(optional)
+
microRNA and reference
gene primer sets
Pick-&-Mix PCR panels
(Ready-to-Use)
Predefined PCR panels
Human, Mouse&Rat, and Focus PCR panels
(Ready-to-Use)
Description page 6,
protocol page 20
Description page 9,
protocol page 43
Description page 7,
protocol page 29+36
+
ExiLENT SYBR® Green master mix
1, 2,5 or 20ml
Figure 1.
Description on page 5
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
I. Reagent kits
Universal cDNA synthesis kit II, 8-64 rxns (product # 203301)
This kit contains all reagents required for first-strand cDNA synthesis for 8-64 reactions1).
The UniSp6 RNA Spike-in template can be used by itself or in combination with the cel-miR39-3p template provided in the RNA Spike-in kit:
Contents
Amount supplied
5× Reaction buffer
128 μL, 5x concentrated
Enzyme mix
64 μL, 10x concentrated
Nuclease free water
1 ml
UniSp6, RNA Spike-in template3)
12 fmol, dried down
Table 1.
miRCURY LNA™ Universal RT microRNA PCR, Starter Kit (product # 203351)
This kit contains all reagents required to perform 20 cDNA reactions of 10 μL volume and
100 PCR reactions. Kit includes primer set for the spike control UniSp6 (included), one
candidate endogenous control primer set (miR-103a-3p) and two validated primer sets of
your choice.
Contents
Amount supplied
5x Reaction buffer2)
128 μL, 5x concentrated
Enzyme mix
24 μL, 10x concentrated
UniSp6, RNA Spike-in template
12 fmol, dried down
UniSp6 RNA Spike-in control primer set v24), lyophilized
200 rxn
hsa-miR-103a-3p primer set (also works for mmu+rno),
lyophilized
200 rxn
2 primer sets free of choice from stocked primers
2x 200 rxn
ExiLENT SYBR® Green master mix, 2x concentrated
500 μL
Nuclease free water
1.25 mL
Table 2.
1) Number of reactions is based on a standard reaction volume of 10 μL to 80 μL. Reaction volume depends on the application
and number of assays to profi le. Please consult Figure 4 for details.
2) Includes universal reverse transcription primer.
3) Used exclusively for the UniSp6 RNA Spike-in control primer set included in the ExiLENT SYBR® Green master mix kit.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
ExiLENT SYBR® Green master mix
upplied
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These kits contain all reagents required for PCR amplification of microRNAs. In addition,
a positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this
kit for amplification of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA
synthesis kit II.
ExiLENT SYBR® Green master mix, 1 ml (product # 203401). The master mix is provided in
an amount sufficient for 200 reactions of 10 μL:
Contents
Amount supplied
ExiLENT SYBR® Green master mix, 2x concentrated
2x 0.5 ml, 2x concentrated
Nuclease free water
1x 1.25 ml
UniSp6 RNA Spike-in control primer set v21), lyophilized
200 rxn
Table 3.
ExiLENT SYBR® Green master mix, 2.5 ml (product # 203403). The master mix is provided
in an amount sufficient for 500 reactions of 10 μL:
Contents
Amount supplied
ExiLENT SYBR® Green master mix, 2x concentrated
2x 1.25 ml, 2x concentrated
Nuclease free water
2x 1.25 ml
UniSp6 RNA Spike-in control primer set v21), lyophilized
200 rxn
Table 4.
ExiLENT SYBR® Green master mix, 20 ml (product # 203421). The master mix is provided in
an amount sufficient for 4000 reactions of 10 μL:
Contents
Amount supplied
ExiLENT SYBR® Green master mix, 2x concentrated
2x 10 ml, 2x concentrated
Nuclease free water
1x 20 ml
UniSp6 RNA Spike-in control primer set v21), lyophilized
200 rxn
Table 5.
1) Used exclusively for detection of the UniSp6 RNA spike-in provided with the Universal cDNA synthesis kit II.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
II. Primer Sets and Panels
MicroRNA LNA™ PCR primer set (product # 204000-206xxx and 2100000-21xxxxx)
LNA™ PCR primer sets are designed for optimal performance with the Universal cDNA
Synthesis Kit II and the ExiLENT SYBR® Green master mix, kit II. The performance of LNA™
primer sets will be affected if used in combination with less than optimal reagents. The
primer sets are supplied in sufficient amounts for 200 reactions of 10 μL.
Contents
LNA™ PCR Primer set (dried down)
Table 6.
Reference gene primer set (product # varies)
The Reference gene primer sets are designed for use with the microRNA primers sets above
as reference genes for normalization. The primer set is supplied in sufficient amount for 200
reactions of 10 μL.
Contents
LNA™ PCR Primer set (dried down)
Table 7.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Pre-defined microRNA PCR Panels (product # varies)
The Human, Mouse&Rat and Focus microRNA PCR panels Ready-to-Use consist of
either 96-well or 384-well PCR plates containing dried down LNA™ primer sets for one
10 μL real-time PCR reaction per well. The LNA™ primer sets are designed for optimal
performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR® Green master
mix, kit. The performance of the LNA™ primer sets will be affected if they are used in
combination with less than optimal reagents.
Contents Human miRNome panel I and II, V4
384-well PCR plates supplied with LNA™ primer sets dried down, one 10 μL reaction per well:
Panel I
Panel II
372 LNA™ primer sets for the
amplification of human microRNAs1)
380 LNA™ primer sets for the
amplifi cation of human microRNAs1)
3 inter-plate calibrators
3 inter-plate calibrators
3 primer sets for reference genes2)
1 blank well
5 RNA Spike-in control primer sets3)
1 blank well
Table 8.
Contents Mouse&Rat miRNome panel I and II, V4
384-well PCR plates supplied with LNA™ primer sets dried down, one 10 μL reaction per well:
Panel I
Panel II
372 LNA™ primer sets for the
amplification of mouse and rat microRNAs1)
380 LNA™ primer sets for the
amplifi cation of mouse and rat microRNAs1)
3 inter-plate calibrators
3 inter-plate calibrators
3 primer sets for reference genes
2)
1 blank well
5 RNA Spike-in control primer sets3)
1 blank well
Table 9.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Contents Serum/Plasma Focus microRNA PCR panel, V4
PCR plates compatible with various real-time PCR instruments are available and supplied
with LNA™ primer sets dried down, one 10 μL reaction per well:
96-well (2 plates)
384-well plate (2 panels per plate)
179 LNA™ primer sets for the amplification
of human microRNAs1+2)
2x179 LNA™ primer sets for the amplification
of human microRNAs1+2)
2x3 Inter-plate calibrators
2x6 Inter-plate calibrators
5 RNA Spike-in control primer sets3)
2x5 RNA Spike-in control primer sets3)
2 blank wells - 1 in each plate
2x2 blank wells
Table 10.
Contents Cancer Focus microRNA PCR panel, V4
PCR plates compatible with various real-time PCR instruments are available and supplied
with LNA™ primer sets dried down, one 10 μL reaction per well:
96-well (1 plates)
384-well plate (4 panels per plate)
84 LNA™ primer sets for the amplification
of human microRNAs1)
3 Primer sets for potential
reference genes2)
4x84 LNA™ primer sets for the amplification
of human microRNAs1)
4x3 Primer sets for potential
reference genes2)
3 Inter-plate calibrators
4x3 Inter-plate calibrators
5 RNA Spike-in control primer sets3)
4x5 RNA Spike-in control primer sets3)
1 blank well
1 blank well
Table 11.
1) Please go to www.exiqon.com/mirna-pcr to download plate layout files.
2) Human Panels and Cancer Focus Panel: Three snRNAs (U6snRNA, SNORD38B, SNORD49A). Mouse&Rat Panels: Three snRNAs
(U6snRNA, RNU5G, RNU1A1). Serum/plasma Focus Panel: miR-103a-3p, miR-191-5p, miR-423-5p, miR-16-5p, miR-425-5p,
miR-93-5p, miR-451a are regarded reference gene candidates.
3) The RNA Spike-in control primer sets targets the UniSp6 RNA spike-in supplied in the Universal cDNA synthesis kit II and the 4 RNA
spike-ins contained in the RNA Spike-in kit (UniSp2, UniSp4, UniSp5, and cel-miR-39-3p).
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Pick-&-Mix microRNA PCR Panel, (product # 203893-203897 and 203818-203819)
The Pick-&-Mix microRNA PCR Panels consist of either 96-well PCR plates or 384-well
PCR plates containing custom selections of dried down microRNA LNA™ PCR primer sets
for one 10 μL real-time PCR reaction per well, ready-to-use. PCR plates compatible with
various real-time PCR instruments are available. The LNA™ primer sets are designed for
optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR® Green
master mix kit. The performance of the LNA™ primer sets will be affected if they are used
in combination with less than optimal reagents.
Contents
PCR plates supplied with customer defined LNA™ primer sets, reference gene primer sets,
and RNA Spike-in control primer sets, dried down, one 10 μL reaction per well1):
96-well PCR plates1)
384-well PCR plates1)
10 primer sets in 8 replicates
22 primer sets in 16 replicates
22 primer sets in 4 replicates
46 primer sets in 8 replicates
92 primer sets in 1 replicate
94 primer sets in 4 replicates
96-well flexible layout
380 primer sets in 1 replicate
384-well flexible layout
Table 12.
1) Please go to www.exiqon.com/pick-and-mix to configure a Pick-&-Mix microRNA PCR Panel.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Storage
All microRNA PCR Panels, LNA™ primer sets and Reference gene primer sets
The PCR panels and primer sets are shipped dried down at room temperature. The primers
can be stored between +4°C and -20°C. Under these conditions, all components are stable
for at least 12 months. Aer resuspension, it is recommended to store LNA™ primer sets
and Reference gene primer sets in aliquots at -20°C to avoid repeated freeze-thaw cycles.
Universal cDNA synthesis kit II and ExiLENT SYBR® Green master mix
These kits are shipped on dry ice in polystyrene containers and should be stored at -15°C
to -25°C. Do not store in a frost-free freezer. Under these conditions, all components are
stable until the expiry date on the package or vial. It is recommended that the RNA spike-in
be stored in aliquots at -20°C aer re-suspension to avoid repeated freeze-thaw cycles.
Additional required materials
Reagents not supplied
• ROX or other passive reference dye (required on some PCR cyclers) see Tip 8 on page 54
for more. See tip 8
Materials and Equipment not supplied
• Nuclease-free PCR tubes or plates for use with individual assays
• Nuclease-free, aerosol barrier pipette tips
• Nuclease-free, low nucleic acid binding microcentrifuge tubes
(e.g. Eppendorf DNA LoBind tubes product and original NUNC vials)
• Sealing foils for PCR plates
• Micro-centrifuge and plate centrifuge
• Heating block, thermal cycler or other incubators
• Real-time PCR instrument
Optional but recommended product
• miRCURY LNA™ Universal RT microRNA PCR, RNA Spike-in kit
(product# 203203)
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Recommended accompanying products
Exiqon recommends the Exiqon GenEx qPCR soware for comprehensive and convenient
data analysis. GenEx includes a wizard for import of Exiqon miRCURY™ Universal RT
microRNA PCR data and offers advanced methods to analyze real-time qPCR data in a few
simple steps. The soware includes tools for selection and validation of reference genes,
data pre-processing and comprehensive statistical analyses. For more information and to
download a free trial, please go to www.exiqon.com/qpcr-soware. The following Exiqon
GenEx products are available:
Exiqon GenEx6 Industrial - Exiqon version of GenEx,
qPCR analysis soware, industrial license
Exiqon GenEx6 Academic - Exiqon version of GenEx,
qPCR analysis soware, academic license
Exiqon recommends the miRCURY™ RNA Isolation kits for purification of total RNA
or small RNA fraction. RNA purified using the miRCURY™ RNA Isolation kits is fully
compatible with the miRCURY LNA™ Universal RT microRNA PCR System. The following
kits are available:
miRCURY™ RNA Isolation Kit – Cell & Plant
Provides a rapid method for purification of total RNA from cultured animal cells, small
tissue samples, blood, bacteria, yeast, fungi and plants.
miRCURY™ RNA Isolation Kit – Tissue
Specifically designed for purification of total RNA from tissue samples.
miRCURY™ RNA Isolation Kit – Biofluids
Kit for purification of low abundant small RNAs from samples such as serum, plasma, urine
and CSF. See tip 2
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Product description
A unique system for microRNA profiling
miRCURY LNA™ Universal RT microRNA PCR offers the best available combination of
performance and ease-of-use on the microRNA real-time PCR market because it unites two
important features (Figure 2):
1. Universal RT – One first-strand cDNA synthesis reaction provides template for all
microRNA real-time PCR assays. This saves precious sample, reduces technical variation,
requires use of less reagents, and saves time in the laboratory.
2. LNA™ PCR amplification – Both PCR amplification primers (forward and reverse) are
microRNA specific and optimized with LNA™. The result is: 1) exceptional sensitivity as well
as extremely low background enabling accurate quantification of very low microRNA levels
and 2) highly specific assays that allow discrimination between closely related microRNA
sequences.
miRCURY LNA™ Universal RT microRNA PCR offers solutions both for high-throughput
microRNA expression profiling and for quantification of individual microRNAs.
Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System.
Step 1: First-strand synthesis (RT)
Mature microRNA
A)
AAAAAAAAAAAAAAAAAAAA
B)
AAAAAAAAAAAAAAAAAAAA
TTTTTTTTTTTTTTT
5’ universal tag
3’ degenerate anchor
Step 2: Real-time PCR amplification
miR-specific forward primer
A)
LNA
LNA
LNA
TTTTTTTTTTTTTTT
LNA
LNA
LNA
miR-specific reverse primer
B)
Figure 2. A poly-A tail is added to the mature microRNA template (step 1A). cDNA is
synthesized using a poly-T primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B).
The cDNA template is then amplified using microRNA-specific and LNA™-enhanced forward
and reverse primers (step 2A). SYBR® Green is used for detection (step 2B).
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Control Assays
There are 3 different types of control assays available in the miRCURY LNA™ Universal RT
microRNA PCR system:
• Reference assays and reference candidates
• Inter-plate calibrators
• RNA spike-in assays
All of these control assays are available in the microRNA PCR panels. One RNA spike-in
template is provided with the Universal cDNA Synthesis Kit II, while the assay that will detect this
RNA spike-in is available in the ExiLENT SYBR® Green master mix. Additionally 4 RNA spike-in
templates are available as a spike-in kit. The assays for detection of these 4 templates as well as
the reference assays are available as individual primer sets.
Reference assays and reference candidates
These assays detect small non-coding RNAs - either small nuclear RNA, small nucleolar RNA or
microRNA – which frequently are found to be stably expressed across different cells or tissues.
Reference assays may therefore be candidate assays for normalization in a profiling study with
several samples. Though this is a good and recommended approach, great caution should be
taken in the selection of reference genes. The danger of using endogenous reference genes lies in
the assumption that a specific gene is expressed at the exact same level in all sample types. This
is rarely true. The selection of reference genes should therefore be made with care, and should
be specific to the sample set you are working with. The actual selection of reference genes to be
used for normalization should always be based on a determination of the most stably expressed
gene(s) which may be done using either GeNorm or NormFinder – both tools that are integrated
within Exiqon’s GenEx data analysis soware. When applicable, we recommend using microRNA
rather than small nuclear RNA or small nucleolar RNA for normalization. Firstly, small nuclear and
nucleolar RNAs are longer RNA species than microRNA and may purify differently from microRNA.
Moreover, small nuclear and nucleolar RNAs have entirely different functions as well as subcellular locations, and finally certain samples like blood plasma does not contain the small nuclear
and nucleolar RNAs. Global mean normalization is a preferred alternative to using reference genes
for normalization when working with panels and samples where many microRNAs are screened
per sample and where many microRNA are called (detected) in all samples. Exiqon’s GenEx will
easily perform global mean normalization. To read more on normalization. See tip 11
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Inter-plate calibrators
Three wells within the pre-defined Human, Mouse&Rat, and Focus PCR Panels contain the
inter-plate calibrator assay (annotated as UniSp3 IPC in the plate layout files). Depending on
the plate layout, the Pick-&-Mix Panels contain at least three inter-plate calibrators. Each
of these wells, contain a pre-aliquoted primer pair and a DNA template and therefore the
variation is very minimal from well-to-well and from plate-to-plate of these assays. The
inter-plate calibrators are used for calibration between PCR plate runs which is very useful
on some instruments that apply the cycle threshold method for Cq determination such as
the ABI7900 PCR cycler. Since the inter-plate calibrators are independent of cDNA quality
in order to give a signal (but may be affected by PCR inhibitors in the sample) they may be
used to quality control each plate run.
Inter-plate calibration (IPC) can easily be performed in the data analysis soware Exiqon’s
GenEx. Alternatively, IPC may be performed manually by using the IPC assay replicates as
follows. For each plate, verify that the replicates have Cq standard deviation within 0.5. If
this is not the case, eliminate the outlier if this can be identified. Calculate the average of
the replicates for each plate, the overall average (average of IPC values from all plates). The
calibration factor is calculated as the difference between plate average and overall average
for each plate (calibration factor = IPCplate-IPCoverall). An example is shown in Table 13.
Finally, calibrate each plate by subtracting the calibration factor from all Cq values in the plate.
Plate 1
Plate 2
Plate 3
let-7c
21.12
20.93
21.34
IPC plate average
19.72
19.70
20.00
IPC overall average
19.81
19.81
19.81
Calibration factor
-0.09
-0.11
0.19
let-7c calibrated
21.21
21.04
21.15
Table 13. Example of inter-plate calibration (IPC)
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
RNA spike-ins (synthetic control templates)
The primary purpose of the RNA spike-ins and the matching primer pairs for detection of
these is to provide controls for the quality of the RNA isolation, the cDNA synthesis reaction
and the PCR. RNA isolations may vary in yield, purity and integrity. Some sample types may
contain compounds that inhibit the cDNA synthesis or the PCR even though the RNA has
been purified using the best standard procedures. This may result in different efficiencies
of the reverse transcription or PCR between compared samples. One way to control for
differences in efficiencies at each experimental level (isolation, cDNA synthesis, and PCR) is
by adding known RNA spike-ins to the sample prior to isolation and cDNA synthesis. Use
of the RNA spike-ins may also reveal if nucleases are present. Aer conducting the PCR but
before initiating the data analysis, wells detecting RNA spike-ins are compared and outlier
samples may be identified and considered for exclusion in the further data analysis.
We have designed a flight of RNA spike-ins for this purpose. The UniSp6 RNA Spike-in
template is provided with the Universal cDNA synthesis kit II. Additionally four RNA spike-in
templates can be obtained with the separate RNA Spike-in kit. Here, a vial of three RNA
spike-in templates, UniSp2, UniSp4 and UniSp5, mixed at different concentrations can be
used during RNA isolation. The cel-miR-39-3p RNA template provided in a separate vial
in the RNA Spike-in kit can be mixed with the UniSp6 template from the Universal cDNA
synthesis kit II to obtain two different template concentrations. This combination can be
added during the cDNA synthesis. Five wells in pre-defined PCR panel plates contain the
matching primer sets. The selection of RNA Spike-in control primer set in the Pick-&-Mix
PCR Panel can be customized to the specific need. A UniSp6 control primer set is also
provided with the ExiLENT SYBR® Green master mix kit, which is to be used with our nonplate based PCR primer set products.The RNA spike-ins are shipped dried down and must
be re-suspended before use.
If the RNA Spike-in kit with multiple RNA spike-ins is used, follow the protocol
accompanying that product.
If UniSp6 is to be used alone:
1. Re-suspend the UniSp6 RNA spike-in by adding 80 μL nuclease free water to the tube.
2. Mix by vortexing and spin down. Leave for 20-30 min. on ice to fully dissolve RNA spikein. Mix by vortexing and spin down. Store in aliquots at -20°C
3. Prior to the RT reaction, add 1 μL synthetic spike-in (108 copies/μL) per 20 ng sample RNA.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This is
only relevant when using individual LNA™ primer sets and Reference gene primer sets in a
multi-plate set-up. The microRNA PCR panels already contain an inter-plate calibrator. Add
1 μL synthetic spike-in (108 copies/μL) to 20 ng of a complex RNA sample
(e.g. total RNA from MS2, yeast, or a cell line; not provided with the kit). Proceed with
first strand synthesis and subsequently real-time PCR as described in the protocols of the
current instruction manual. At least one spike-in amplification reaction per PCR plate is
used for inter-plate calibration. See tip 9
Experimental design
Before starting the experiment, it is essential to consider the experimental setup and
consider the number of replicates needed for obtaining significant results – replicates being
technical as well as biological. The number of biological replicates required varies from
experiment to experiment depending on the variation within and between the groups. We
recommend that a No Template Control (NTC) is included in the study every time a new
experiment is set up, to set the background level. The most optimal NTC is a mock up
sample preparation including only carrier RNA as sample. The NTC should be run on all
assays included in the study. We define the background of an assay as 5 Cq values below
the NTC level. Furthermore we recommend including spike-ins found in the Spike-in kit to
provide full quality control over all steps in the profiling (see figure 3). Finally it is necessary
to include a number of candidate reference miRNAs, which are expected to be constitutively
expressed across the different experimental conditions, for data normalization.
Experimental design
Comes with
Universal cDNA
synthesis kit II
Sold as separate Spike-in kit (203203)
UniSp2
UniSp4
UniSp5
cel-miR-39-3p
UniSp6
Included in all
panel products
UniSp3
Assays available as
individual assays or in
ready-to-use panels
miR-A
miR-X
Sample n
Sample n+1
Sample preparation evaluation
cDNA synthesis evaluation
NTC
Figure 3.
16
Inter plate Calibration
Sample profiling
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Before starting the Experiment
Important note
RNA work requires specific handling and precautions to prevent RNase
contamination of the reagents and degradation of the RNA sample. Find information
on how to handle RNA in the tips section starting on page 52. The tips section
also provides simple guidelines for good laboratory practice to ensure optimal
performance of PCR experiments.
Before setting up a real-time PCR experiment, there are a number of practical experimental
design parameters that should be considered:
RNA input - The miRCURY LNA™ Universal RT microRNA PCR protocol is optimized
for use of 20 ng total RNA per 20μL cDNA synthesis reaction. The exact amount of total
RNA needed depends on whether the downstream application is individual assays or
panels. Furthermore, the amount of total RNA to be used may also vary depending on the
microRNA expression levels in the cells or tissue to be analyzed. For highly expressed
microRNAs it is possible to use down to 10 pg total RNA as starting material. For weakly
expressed microRNAs it may be possible to use up to 200 ng of total RNA; however, in
samples with high amounts of PCR inhibitors (e.g. FFPE tissue samples), this may not
be feasible. Finally, inhibitors may be present in RNA preparations from certain samples
e.g. serum and plasma. Prior to conducting a larger microRNA profiling study, it is
recommended to optimize the amount of input RNA to the RT reaction in order to avoid
conducting a larger study where inhibition occurs sporadically throughout the data set.
Information on how to extract and handle RNA can be found in the tips section. In short,
total RNA should be prepared using a method that preserves small RNA species. DNase
treatment may be necessary. When using commercially available kits, please ensure that
the total RNA preparation is guaranteed to contain microRNAs.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Note: Blood serum and plasma are particular sample types that require special RNA
purification procedures and the amount of RNA present in the samples can usually not be
accurately determined. Due to the low levels of microRNAs and potentially high levels of
inhibitors in samples derived from serum and plasma, specific recommendations for how
to set up experiments using these types of sample can be found in the miRCURY LNA™
Universal RT microRNA PCR, Instruction Manual “Biofluid samples”.
Normalization – when running individual assays or when configuring a Pick-&-Mix
microRNA PCR panel it is important to consider how the data will be normalized. For tips
and recommendations on choosing the correct reference genes and how to test and validate
reference genes, please see section on reference assays (page13) and the Tip 11 (page 58).
See tip 11
Excess volumes required for pipetting – Liquid handling with pipettes or pipetting robots
require excess volumes of reagents due to loss during pipetting. The loss depends on the
available pipetting system but losses in the range from 10% -25% are not uncommon.
All protocols in the current instruction manual reflect the required reaction volumes and
pipetting volumes should be adjusted according to accommodate the pipetting loss of the
available pipetting system.
ROX – ROX is a passive reference dye used by some PCR cycles to obtain a robust read
over the entire array of wells in a 96- or 384-well PCR plate. The requirement for ROX
is instrument dependent and we recommend to follow the instrument manufactures
guidelines on this. See tip 8
ABI instruments – the default settings on ABI real-time PCR cyclers are not suitable for
running miRCURY LNA™ Universal RT microRNA PCR. Settings need to be changed from
automatic to manual background and threshold settings to obtain valid PCR data (see
also Tip 10). Furthermore, if the dataset is to be analyzed using the GenEx soware, it is
important that the experiment is set up as an AQ experiment, not RQ. To ensure correct
settings, download the instrument settings file at www.exiqon.com/sds. See tip 10
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
RNA spike-ins – consider how the RNA spike-ins should be applied in the planned study.
Please consult the section on RNA spike-ins on page 15.
Protocols for the first-strand cDNA synthesis and real-time amplication follows on the
next pages:
A. Individual assays, please go to page 20
B. Human and Mouse&Rat microRNA PCR Panels, please go to page 29
C. Focus microRNA PCR Panels, please go to page 36
D. Pick-&-Mix microRNA PCR Panels, please go to page 43
Figure 4 gives an overview and helps identifying which protocol to follow as well as the
recommended cDNA reaction volume needed for a given sample and assay type.
Protocol overview
miRNome
panel I
miRNome
panel I+II
Universal cDNA
reactions per kit
32
Universal cDNA
reaction volume
20 µL
Dilution of cDNA
in ExiLENT
Master Mix
100x
1-96
97-192
Individual
assays
(<96)
16
64
32
64
40 µL
10 µL
20 µL
10 µL
Pick-&-Mix/Focus
80x
Use standard printed manual or download from
www.exiqon.com/pcr-manual
Figure 4.
19
Use biofluids manual from
www.exiqon.com/pcr-manual-biofluids
Cells or tissue
Panels or
primer sets:
Serum plasma or other biofluids
Sample type
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol A - Individual assays
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR,
using the individual assays for human, mouse and rat (product numbers 204000-206xxx).
If working with serum plasma samples or other biofluids, please refer to the specific
miRCURY LNA™ Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Before using the LNA™ PCR primer set , the UniSp6 RNA spike-in control primer set or
the Reference gene primer set for the first time, the primers must be re-suspended:
• Re-suspend the primer set by adding 220 μL nuclease free water to the tube. Mix by
vortexing and spin down. Leave on ice for 20-30 minutes.
Protocol A.
Individual assays
Additional required materials:
• 96- or 384-well plate real-time PCR cycler
• Thermocycler for first-strand cDNA synthesis
• 96/384-well plates or tube strips compatible with available real-time PCR cycler
• Micro centrifuge
• Swing bucket centrifuge for 96-/384-well plates
20
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Checklist:
• Have you considered excess volumes required for using liquid handling robotics? – see page 18
• Did you consider how to use the RNA spike-ins? – please see page 15
• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference
dye. Please follow instrument manufactures recommendations
• ABI instruments: The use of manual background and threshold settings is necessary
for obtaining correct PCR data. Make sure to have the optimal settings by downloading
the instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be
analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ
• Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Protocol A.
Individual assays
21
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Workflow for individual primer sets (per sample)
Phase I: Prepare RNA sample
See page 52 for recommendations
Phase II: cDNA synthesis
See protocol page 23.
LNATM primer sets
Relative expression (log2)
Protocol A.
Individual assays
Phase III: real-time PCR amplification
See protocol page 25.
- Prepare adequate amount of pre-mixed primer
+ PCR Master mix and distribute into wells
- Add cDNA to all primer sets to be analyzed
4
3
2
1
0
-1
Normal
Tumor Total
œ miR-21
œ let-7a
22
Tumor
Tumor
stroma
Phase IV: Data analysis
See data analysis guide online
- Export data for further analysis
- Data pre-processing, normalization
and statistical analysis
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol
The miRCURY LNA™ Universal RT microRNA PCR protocol is a two-part protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-11)
Important: Keep reagents and reactions on ice (or at 4˚C) at all times.
First strand synthesis
Step 1
Dilute template RNA
Adjust each of the template RNA samples to a concentration of
5 ng/μL using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA
spike-ins according to the appropriate RNA Spike-ins protocol (see
page 15), leave on ice for 15-20 minutes. Immediately before use,
remove the Enzyme mix from the freezer, mix by flicking the tubes and
place on ice. Spin down all reagents.
Protocol A.
Individual assays
23
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 3
Combine reagents
according to Table 14
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume
If performing first-strand cDNA synthesis on multiple RNA samples, it
is recommended to prepare an RT working solution of the 5x Reaction
buffer, water, Enzyme mix and RNA spike ins (in the proportion
indicated in the first four lines of Table 14).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Reagent
Volume (μL), RT reaction
5x Reaction buffer
2
Nuclease-free water
4.5
Enzyme mix
1
Synthetic RNA spike ins, optional
replace with H2O if omitted
0.5
Template total RNA (5 ng/μL)
2
Total volume
10
Protocol A.
Individual assays
Table 14. Reverse transcription reaction setup
Step 4
Mix and spin
reagents
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
Step 5
Incubate and heat
inactivate1)
• Incubate for 60 min at 42˚C.
• Heat-inactivate the reverse transcriptase
for 5 min at 95˚C.
• Immediately cool to 4°C.
• Store at 4°C or freeze.
1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks (optional store at 4˚C for up
to 4 days). It is recommended that synthesized cDNA is stored in “low-nucleic acid binding” tubes or plates.
24
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
qPCR protocol
Step 6
Prepare reagents for
real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master mix on
ice and thaw for 15-20 min. Protect the PCR Master mix vials from light.
Immediately before use, mix the PCR Master mix by pipetting up and
down. The rest of the reagents are mixed by vortexing and spun down.
Step 7
Dilute cDNA template
80x in nuclease
free water2)
Immediately before use, dilute only the amount of cDNA template
needed for the planned real-time PCR reactions 80x in nuclease free
water (e.g. add 395 μL nuclease free water to each 5 μL of reaction). It
is important that “low-nucleic acid binding” tubes or plates are used. It
is not recommended to store the 1:80 dilution of cDNA.
Recommendation: Include a passive reference dye in the cDNA
dilution if advised by instrument manufacturer. Please note that the
PCR Master mix does not include ROX. The amount of ROX required is
instrument dependent and it is important to refer to the manufacturer’s
recommendations when deciding how much ROX to use.
See tip 8
2)
Adjust volumes to accommodate your in-house liquid handling system volume loss when pipetting
Protocol A.
Individual assays
25
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 8
Combine PCR Master
mix, PCR primer set
and cDNA according
to Table 15
Mix thoroughly
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume
When multiple real-time PCR reactions are performed with the same
microRNA primer set, it is recommended to prepare a primer master
mix working-solution of the PCR primers and the PCR Master mix (in
the proportion indicated in Table 15).
The following procedure is recommended:
1. Prepare the required amount of primer:master mix workingsolution (see Table 3) and place it on ice. It is recommended to
include excess of all reagents in the master mix to compensate for
pipetting excess material.
2. Place the relevant volume of primer:master mix working-solution
in PCR tubes/wells (see Table 15) and spin tubes/plate briefly in a
centrifuge (1500g for 1 minute), to remove air bubbles.
3. Add cDNA template to each tube/well.
Volume (μL), 96/384-well
plate, tubes or strips
Reagent
PCR Master mix
PCR primer set
5
4)
1
Diluted cDNA template
4
Total volume
10
Table 15. Real-time PCR reaction, pr. 10 μL reaction3)
Protocol A.
Individual assays
Step 9
Mix and spin
reagents
Mix the reaction by gentle pipetting to ensure that all reagents are
mixed thoroughly. After mixing cap tubes or strips or seal the plate
with optical sealing as recommended by the manufacturer. Spin down
in a centrifuge (1500g for 1 minute). The experiment can be paused at
this point. Store the reactions protected from light at 4°C for up to 24
hours.
3) If using a 96-well cycler with a minimum recommended volume of 20 μL (as some ABI instruments), then use 10 μL reaction volume
and set the instrument settings at 20 μL.
4) The PCR primer set must be dissolved prior to real-time PCR set-up, see page 20.
26
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
ABI instrument user?
Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file
Step 10
Real-time PCR
amplification
Perform real-time PCR amplification followed by melting curve
analysis according to Table 16.
Process step
Polymerase Activation/
Denaturation
Amplification
Melting curve analysis7)
Settings, LC480
instrument5)
95˚C, 10 min
Settings, other
instruments3)
95˚C, 10 min
45 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s6)
Optical read
Yes
40 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s6)
Optical read
Yes
Table 16. Real-time PCR cycle conditions
Step 11
Analyze data
For tips on normalization, please see Tip 11, page 58. We recommend
performing normalization and further data analysis with the Exiqon
GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis).
Please refer to our data analysis guide
for recommendations.
See tip 11
27
Protocol A.
Individual assays
Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that it
is not recommended to use auto Ct settings. Furthermore, if the data is
to be analyzed using Exiqon GenEx, the experiment must be set up as
an AQ experiment, not RQ. For a guide on how to set manual baseline
and threshold, refer to Tip 10, page 56 in the tips section. If you are
using a Roche LC480 instrument, we recommend analysis using the
2nd derivative method.
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol A.
Individual assays
5) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up
to 40.
6) The ramp-rate of cooling from 95˚C to 60˚C should be set to 1.6°C/s. This is equivalent to 100% under standard cycling conditions on
the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised.
7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction. Melting
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier.
Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
28
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol B - Human and Mouse&Rat
microRNA PCR Panels
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR
using the following products:
• Human miRNome PCR Panel (product numbers 203611 to 203618)
• Mouse&Rat miRNome PCR Panel (product numbers 203709 to 203716)
If working with serum plasma samples or other biofluids, please refer to the specific
miRCURY LNA™ Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Additional required materials:
• 384-well plate real-time PCR cycler
• Thermocycler for first-strand cDNA synthesis
• Micro centrifuge
• Tube for mixing water and master mix (10 ml)
• Sealing foils for PCR plates
• Swing bucket centrifuge for 96/384-well plates
• Recommended: Liquid handling robot for pipetting
29
Protocol B. Human and
Mouse&Rat microRNA PCR Panels
Checklist:
• Have you considered excess volumes required for using liquid handling robotics? –
see page 18
• Did you consider how to use the RNA spike-ins? – please see page 15
• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference
dye. Please follow instrument manufactures recommendations
• ABI instruments: The use of manual background and threshold settings is necessary for
obtaining correct PCR data. Make sure to have the optimal settings by downloading the
instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed
using GenEx, the experiment must be set up as an AQ experiment, not RQ.
• Have you optimized the input amount to the RT reaction in order to avoid inhibition?
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Workflow for Human and Mouse&Rat microRNA PCR Panels (per sample)
Phase I: Prepare RNA sample
See page 52 for recommendations
Phase II: cDNA synthesis
See protocol page 31.
Relative expression (log2)
Protocol B. Human and
Mouse&Rat microRNA PCR Panels
Phase III: real-time PCR amplification
See protocol page 33.
- Mix cDNAs with PCR Master mix
- Add cDNA:PCR Master mix to
PCR plates
4
3
2
1
0
-1
Normal
Tumor Total
œ miR-21
œ let-7a
30
Tumor
Tumor
stroma
Phase IV: Data analysis
See data analysis guide online
- Export data for further analysis
- Data pre-processing, normalization
and statistical analysis
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol
The miRCURY LNA™ Universal RT microRNA PCR protocol is a two-part protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-9)
Important: Keep reagents and reactions on ice (or at 4˚C) at all times.
First strand synthesis:
Step 1
Dilute template RNA
Adjust each of the template RNA samples to a concentration of 5 ng/μl
using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA
spike-in(s) according to the appropriate RNA Spike-in protocol (see
page 15), leave on ice for 15-20 minutes. Immediately before use,
remove the Enzyme mix from the freezer, mix by flicking the tubes and
place on ice. Spin down all reagents.
Protocol B. Human and
Mouse&Rat microRNA PCR Panels
31
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 3
Combine reagents
according to Table 17
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume.
If performing first-strand cDNA synthesis on multiple RNA samples, it
is recommended to prepare an RT working solution of the 5x Reaction
buffer, water, Enzyme mix and RNA spike ins (in the proportions
indicated in the first four lines of Table 17).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
5x Reaction buffer
Panel I
Volume
(μL)
4
Panel I+II
Volume
(μL)
8
Nuclease-free water
9
18
Enzyme mix
2
4
Synthetic RNA spike ins, optional
replace with H2O if omitted
Template total RNA (5ng/μL)
1
2
4
8
Total volume
20
40
Reagent
Protocol B. Human and
Mouse&Rat microRNA PCR Panels
Table 17. Reverse transcription reaction setup
Step 4
Mix and spin
reagents
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
Step 5
Incubate and heat
inactivate1)
• Incubate for 60 min at 42°C.
• Heat-inactivate the reverse transcriptase
for 5 min at 95°C.
• Immediately cool to 4°C.
• Store at 4°C or freeze.
1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks (optional store at 4˚C for up
to 4 days). It is recommended that synthesized cDNA is stored in “low-nucleic acid binding” tubes or plates.
32
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
qPCR protocol:
Step 6
Prepare reagents for
real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master mix
on ice and thaw for 15-20 min. Protect the PCR Master mix vials from
light. Immediately before use, mix the PCR Master mix by pipetting up
and down. The rest of the reagents are mixed by vortexing and spun
down.
Step 7
Combine PCR Master
mix, water and cDNA
and add to PCR plates2)
The following procedure is recommended to avoid low concentrations
of cDNA from adhering to tube surface:
1. Before removing the plate seal, briefly spin
down the plate(s) in a plate centrifuge.
2. Combine 2x PCR Master mix and water. Panel I: 2000 μL 2x master
mix and 1980 μL water, Panel I+II: 4000 μL 2x master mix and 3960
μL water.
3. Mix gently and spin down.
4. Add 20 μL cDNA (panel I) or 40 μL cDNA (panel I+II) and mix.
5. Add 10 μL PCR Master mix: cDNA
mix to each well3).
6. Seal the plate with optical sealing as recommended by the
instrument manufacturer.
7. Spin plate briefly in a plate centrifuge (1500g for
1 minute), to to collect the sample.
Mix thoroughly
The experiment can be paused at this point. Store the reactions
protected from light at 4°C for up to 24 hours.
See tip 8
33
Protocol B. Human and
Mouse&Rat microRNA PCR Panels
Recommendation: Include a passive reference dye in the cDNA
dilution if advised by instrument manufacturer. Please note that the
PCR Master mix does not include ROX. The amount of ROX required is
instrument dependent and it is important to refer to the manufacturer’s
recommendations when deciding how much ROX to use.
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
ABI instrument user?
Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file
Step 8
Real-time PCR
amplification
Perform real-time PCR amplification followed by melting curve analysis
according to Table 18.
Process step
Polymerase Activation/
Denaturation
Amplification
Melting curve analysis6)
Settings, LC480
instrument 4)
95˚C, 10 min
Settings, other
instruments
95˚C, 10 min
45 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s5)
Optical read
40 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s5)
Optical read
Yes
Yes
Table 18. Real-time PCR cycle conditions
Protocol B. Human and
Mouse&Rat microRNA PCR Panels
Step 9
Analyze data
Perform initial data analysis using the software supplied with the
real-time PCR instrument to obtain raw Cq values (Cp or Ct, depending
on PCR instrument). If you are using an ABI instrument, please note
that it is not recommended to use auto Ct settings. For a guide on how
to set manual baseline and threshold, refer to Tip 10, page 56 in the
tips section. Furthermore, if the data is to be analyzed using Exiqon
GenEx, the experiment must be set up as an AQ experiment, not
RQ. Alternatively, use ABI settings files available from www.exiqon.
com/sds. If you are using a Roche LC480 instrument, we recommend
analysis using the 2nd derivative method.
For tips on normalization, please see Tip 11, page 58. We recommend
performing normalization and further data analysis with the Exiqon
GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis).
Please refer to our data analysis guide for recommendations.
See tip 11
34
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
2) Adjust volumes to accommodate your in-house liquid handling system and the inaccuracy these have when pipetting.
3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.
4) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up
to 40.
5) The ramp-rate of cooling from 95˚C to 60˚C should be set to 1.6°C/s. This is equivalent to 100% under standard cycling conditions on
the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised.
6) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction. Melting
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier. Note:
The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
Protocol B. Human and
Mouse&Rat microRNA PCR Panels
35
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol C - Focus microRNA
PCR Panels
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR,
using the Cancer Focus microRNA PCR panels or any of the Toxicology Focus microRNA
PCR Panels, 96-well plates and 384-well plates.
If working with serum plasma samples or other biofluids, please refer to the specific
miRCURY LNA™ Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Protocol C.
Focus microRNA PCR Panels
Additional required materials:
• 96- or 384-well plate real-time PCR cycler
• Thermocycler for first-strand cDNA synthesis
• Micro centrifuge
• Tube for mixing water and master mix (10 ml)
• Sealing foils for PCR plates
• Swing bucket centrifuge for 96/384-well plates
• Recommended: Liquid handling robot for pipetting
Checklist:
• Have you considered excess volumes required for using liquid handling robotics? – see page 18
• Did you consider how to use the RNA spike-ins? – please see page 15
• ROX: The ExiLENT SYBR® Green master mix does not include the ROX passive reference dye.
Please follow instrument manufactures recommendations
• ABI instruments: The use of manual background and threshold settings is necessary for
obtaining correct PCR data. Make sure to have the optimal settings by downloading the
instrument settings file at www.exiqon.com/sds. Furthermore, if the data is to be analyzed
using GenEx, the experiment must be set up as an AQ experiment, not RQ
• Have you optimized the input amount to the RT reaction in order to avoid inhibition?
36
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Workflow for Focus microRNA PCR Panels (per sample)
Phase I: Prepare RNA sample
See page 52 for recommendations
Phase II: cDNA synthesis
See protocol page 38.
4
3
2
1
0
-1
Normal
Tumor Total
œ miR-21
œ let-7a
37
Tumor
Tumor
stroma
Phase IV: Data analysis
See data analysis guide online
- Export data for further analysis
- Data pre-processing, normalization
and statistical analysis
Protocol C.
Focus microRNA PCR Panels
Relative expression (log2)
Phase III: real-time PCR amplification
See protocol page 40.
- Mix cDNAs with PCR Master mix
- Add cDNA:PCR Master mix to
PCR plates
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol
The miRCURY LNA™ Universal RT microRNA PCR protocol is a two-part
protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-10)
Important: Keep reagents and reactions on ice (or at 4˚C) at all times.
First strand synthesis:
Adjust each of the template RNA samples to a concentration of 5 ng/
μL using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA
spike-in(s) according to the appropriate RNA Spike-in protocol (see
page 15), leave on ice for 15-20 minutes. Immediately before use,
remove the Enzyme mix from the freezer, mix by flicking the tubes and
place on ice. Spin down all reagents.
Protocol C.
Focus microRNA PCR Panels
Step 1
Dilute template RNA
38
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 3
Combine reagents
according to Table 19
Note: remember to
calculate necessary
excess volume for
pipetting and robotic
dead volume
When performing first-strand cDNA synthesis on multiple RNA
samples, it is recommended to prepare an RT working solution of
the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the
proportions indicated in the first four lines of Table 19).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Reagent
Per panel Volume (μL)
5x Reaction buffer
2
Nuclease-free water
4.5
Enzyme mix
1
Synthetic RNA spike ins, optional
replace with H2O if omitted
Template total RNA (5ng/μL)
0.5
Total volume
10
2
Table 19. Reverse transcription reaction setup
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
Step 5
Incubate and
heat inactivate1)
• Incubate for 60 min at 42˚C.
• Heat-inactivate the reverse transcriptase
for 5 min at 95˚C.
• Immediately cool to 4°C.
• Store at 4°C or freeze.
1) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks (optional store at 4˚C for up
to 4 days). It is recommended that synthesized cDNA is stored in “low-nucleic acid binding” tubes or plates.
39
Protocol C.
Focus microRNA PCR Panels
Step 4
Mix and spin
reagents
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
qPCR protocol:
Step 6
Prepare reagents
for real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master mix
on ice and thaw for 15-20 min. Protect the PCR Master mix vials from
light. Immediately before use, mix the PCR Master mix by pipetting up
and down. The rest of the reagents are mixed by vortexing and spun
down.
Step 7
Combine PCR Master
mix, water and cDNA
and add to PCR plates2)
The following procedure is recommended to avoid low concentrations
of cDNA from adhering to tube surface:
1. Before removing the plate seal, briefly spin down the plate(s) in a
plate centrifuge.
2. Combine 2x PCR Master mix and water: 1000 μL 2x master mix and
990 μL water
(Focus panel consisting of 2x 96 assays)
3. Mix gently and spin down.
4. Add 10 μL cDNA (Focus panel consisting of 2x 96 assays).
5. Add 10 μL PCR Master mix: cDNA mix to each well3).
6. Seal the plate with optical sealing as recommended by the
instrument manufacturer.
Mix thoroughly
Protocol C.
Focus microRNA PCR Panels
The experiment can be paused at this point. Store the reactions
protected from light at 4°C for up to 24 hours.
Recommendation: Include a passive reference dye in the cDNA
dilution if advised by instrument manufacturer. Please note that the
PCR Master mix does not include ROX. The amount of ROX required is
instrument dependent and it is important to refer to the manufacturer’s
recommendations when deciding how much ROX to use.
See tip 8
2) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.
3) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.
40
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
ABI instrument user?
Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file
Step 8
Spin plate
Spin plate briefly in a plate centrifuge (1500g for 1 minute), to collect
the sample.
Step 9
Real-time PCR
amplification
Perform real-time PCR amplification followed by melting curve analysis
according to Table 20.
Process step
Polymerase
Activation/Denaturation
Amplification
Melting curve analysis7)
Settings, LC480 Settings, other
instrument5)
instruments 4)
95˚C, 10 min
95˚C, 10 min
45 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s6)
Optical read
Yes
40 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s6)
Optical read
Yes
Table 20. Real-time PCR cycle conditions
41
Protocol C.
Focus microRNA PCR Panels
4) If using a 96-well cycler with a minimum recommended volume of 20 μL (like some ABI instruments), then use 10 μL reaction volume
and set the instrument settings at 20 μL.
5) Five additional amplification cycles is required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40.
6) The ramp-rate of cooling from 95˚C to 60˚C should be set to 1.6°C/s. This is equivalent to 100% under standard cycling conditions on
the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised.
7) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction. Melting
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier. Note:
The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 10
Analyze data
Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that it
is not recommended to use auto Ct settings. Furthermore, if the data is
to be analyzed using Exiqon GenEx, the experiment must be set up as
an AQ experiment, not RQ. For a guide on how to set manual baseline
and threshold, refer to Tip 10, page 56 in the tips section. If you are
using a Roche LC480 instrument, we recommend analysis using the
2nd derivative method.
For tips on normalization, please see Tip 11, page 58. We recommend
performing normalization and further data analysis with the Exiqon
GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis).
Please refer to our data analysis guide for recommendations.
Protocol C.
Focus microRNA PCR Panels
See tip 11
42
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol D - Pick-&-Mix microRNA PCR
Panels
This protocol is used for conducting the first-strand cDNA synthesis and real-time PCR,
using the Pick-&-Mix microRNA PCR Panel, 96-well plates (product number 203893203897) and 384-well plates (product number 203818-203819).
If working with serum plasma samples or biofluid samples, please refer to the specific
miRCURY LNA™ Universal RT microRNA PCR Instruction Manual for biofluid samples at
www.exiqon.com/serum-plasma-pcr-manual.
Additional required materials:
• 96- or 384-well plate real-time PCR cycler
• Thermocycler for first-strand cDNA synthesis
• Micro centrifuge
• Tube for mixing water and master mix (10 ml)
• Swing bucket centrifuge for 96/384-well plates.
• Sealing foils for PCR plates
• Recommended: Liquid handling robot for pipetting
43
Protocol D. Pick-&-Mix
microRNA PCR Panels
Checklist:
• Have you considered excess volumes required for using liquid handling robotics? – see page 18
• Did you consider how to use the RNA spike-ins? – please see page 15
• ROX: The ExiLENT SYBR® Green master mix does not include the ROX passive reference dye. Please
follow instrument manufactures recommendations
• ABI instruments: The use of manual background and threshold settings is necessary for obtaining
correct PCR data. Furthermore, if the data is to be analyzed using GenEx, the experiment must be
set up as an AQ experiment, not RQ. Make sure to have the optimal settings by downloading the
instrument settings file at www.exiqon.com/sds.
• Have you optimized the input amount to the RT reaction in order to avoid inhibition?
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Workflow for Pick-&-Mix PCR plates (per sample)
Phase I: Prepare RNA sample
See page 52 for recommendations
Phase II: cDNA synthesis
See protocol page 46.
Relative expression (log2)
Protocol D. Pick-&-Mix
microRNA PCR Panels
Phase III: real-time PCR amplification
See protocol page 48.
- Mix cDNAs with PCR Master mix
- Add cDNA:PCR Master mix to primer
sets replicates (indicated by colored boxes)
4
3
2
1
0
-1
Normal
Tumor Total
œ miR-21
œ let-7a
44
Tumor
Tumor
stroma
Phase IV: Data analysis
See data analysis guide online
- Export data for further analysis
- Data pre-processing, normalization
and statistical analysis
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
22 microRNAs x 16 samples
Pick-&-Mix microRNA PCR Panel layouts
Overview of the pre-defined plate layouts
available for 96-well and 384-well plates of the
Pick-&-Mix Panel. Wells in dark gray and light
gray are pre-occupied by interplate-calibrators
(UniSp3 IPC) and RNA spike-in controls
(UniSp6), respectively.
1
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1
2
3
4
5
6
7
8
22
CP
IPC
B
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
D
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
9
10
11
12
13
14
15
16
17
18
19
20
21
E
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
F
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
G
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
I
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
J
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
K
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
O
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
P
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CP
IPC
46 microRNAs x 8 samples
10 microRNAs x 8 samples
A
2
A
1
2
3
4
5
6
7
8
9 10 11 12
1
2
3
4
5
6
7
8
9
10
CP
1
IPC
B
1
2
3
4
5
6
7
8
9
10
CP
IPC
C
1
2
3
4
5
6
7
8
9
10
CP
IPC
D
1
2
3
4
5
6
7
8
9
10
CP
IPC
E
1
2
3
4
5
6
7
8
9
10
CP
F
1
2
3
4
5
6
7
8
9
10
CP
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A
1
2
3
4
5
6
7
8
9
B
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CP
D
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
IPC
E
F
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
42
18
19
21
45
20
21
44
45
22
46
22
CP
IPC
23
CP
47
IPC
33
34
35
36
37
38
39
40
41
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CP
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
IPC
IPC
I
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CP
33
34
35
36
37
38
39
40
41
47
IPC
IPC
K
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CP
L
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
IPC
M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CP
47
IPC
G
1
2
3
4
5
6
7
8
9
10
CP
IPC
H
1
2
3
4
5
6
7
8
9
10
CP
IPC
N
25
26
27
28
29
30
31
32
42
43
43
44
44
45
45
46
23
47
4
42
43
20
44
28
32
42
19
43
3
31
32
17
27
30
31
16
2
29
30
15
26
28
29
14
1
27
28
13
25
26
27
12
H
25
26
11
G
J
25
10
46
33
34
35
36
37
38
39
40
41
O
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CP
P
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
46
47
IPC
94 microRNAs x 4 samples
22 microRNAs x 4 samples
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1
2
3
4
5
6
7
8
9 10 11 12
A
1
1
2
2
3
3
4
4
9
10
10
A
B
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
1
2
3
4
5
6
7
8
9
C
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
23
23
24
24
B
13
14
15
16
17
18
19
20
D
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
23
23
24
24
E
25
25
26
26
27
27
28
28
29
29
30
30
31
31
32
32
33
33
34
34
CP
CP
36
C
1
2
3
4
5
6
7
8
F
25
25
26
26
27
27
28
28
29
29
30
30
31
31
32
32
33
33
34
34
CP
CP
36
36
37
37
38
38
39
39
40
40
41
41
42
42
43
43
44
44
45
45
46
46
47
47
48
48
D
G
13
H
37
37
38
38
39
39
40
40
41
41
42
42
43
43
44
44
45
45
46
46
47
47
48
48
14
15
16
17
18
19
20
10
11
21
22
23
CP
9
10
11
IPC
21
22
IPC
23
CP
E
1
2
3
4
5
6
7
8
9
10
11
IPC
F
13
14
15
16
17
18
19
20
21
22
23
CP
1
2
3
4
5
6
7
8
9
10
11
IPC
H
13
14
15
16
17
18
19
20
21
22
23
CP
6
6
7
7
8
8
9
11
11
12
12
36
I
49
49
52
53
53
54
54
56
57
57
58
58
J
49
49
50
50
51
51
52
52
53
53
54
54
55
55
56
56
57
57
58
58
59
59
60
60
K
61
61
62
62
63
63
64
64
65
65
66
66
67
67
68
68
69
69
70
70
IPC
IPC
72
72
50
50
51
51
52
55
55
56
59
59
60
60
L
61
61
62
62
63
63
64
64
65
65
66
67
67
67
68
68
69
69
70
70
IPC
IPC
72
72
M
73
73
74
74
75
75
76
76
77
77
78
78
79
79
80
80
81
81
82
82
83
83
84
84
N
73
73
74
74
75
75
76
76
77
77
78
78
79
79
80
80
81
81
82
82
83
83
84
84
O
85
85
86
86
87
87
88
88
89
89
90
90
91
91
92
92
93
93
94
94
95
95
96
96
P
85
85
86
86
87
87
88
88
89
89
90
90
91
91
92
92
93
93
94
94
95
95
96
96
380 microRNAs x 1 sample
92 microRNAs x 1 sample
1
A
5
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1
2
3
4
5
6
7
8
9 10 11 12
A
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
B
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1
2
3
4
5
6
7
8
9
C
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
D
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
IPC
10
11
IPC
B
13
14
15
16
17
18
19
20
21
22
23
CP
C
25
26
27
28
29
30
31
32
33
34
35
IPC
D
37
38
39
40
41
42
43
44
45
46
47
E
49
50
51
52
53
54
55
56
57
58
F
61
62
63
64
65
66
67
68
69
G
73
74
75
76
77
78
79
80
81
H
85
86
87
88
89
90
91
92
93
E
100
101
102
103
104
105
106
112
113
114
115
116
117
118
F
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
G
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
IPC
H
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
IPC
I
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
59
60
J
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
70
71
72
82
83
84
94
95
96
K
45
97
241
98
242
99
243
249
250
107
108
109
110
111
259
119
24
263
120
244
245
246
247
248
251
252
253
254
255
256
257
258
260
261
262
L
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
CP
M
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
N
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
264
O
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
P
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
IPC
Protocol D. Pick-&-Mix
microRNA PCR Panels
G
5
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Protocol
The miRCURY LNA™ Universal RT microRNA PCR protocol is a two-part protocol consisting of:
1. First-strand cDNA synthesis (Step 1-5)
2. Real-time PCR amplification (Step 6-11)
Important: Keep reagents and reactions on ice (or at 4˚C) at all times.
First strand synthesis:
Adjust each of the template RNA samples to a concentration of 5 ng/
μL using nuclease free water.
Step 2
Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and
immediately place on ice. Mix by vortexing. Re-suspend the RNA
spike-in(s) according to the appropriate RNA Spike-in protocol (see
page 15), leave on ice for 15-20 minutes. Immediately before use,
remove the Enzyme mix from the freezer, mix by flicking the tubes and
place on ice. Spin down all reagents.
Protocol D. Pick-&-Mix
microRNA PCR Panels
Step 1
Dilute template RNA
46
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 3
Combine reagents
according to Table 21
When performing first-strand cDNA synthesis on multiple RNA
samples, it is recommended to prepare an RT working solution of
the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the
proportions indicated in the first four lines of Table 21).
The following procedure is recommended:
1. Prepare the required amount of RT working solution and place it on ice.
2. Dispense RT working solution into nuclease free tubes.
3. Dispense template RNA in each tube.
Reagent
Volume (μL)
< 100 miRNA
analyzed per
sample
Volume (μL)
> 100 miRNA
analyzed per
sample
5x Reaction buffer
2
4
Nuclease-free water
4.5
9
Enzyme mix
1
2
Synthetic RNA spike ins,
optional replace with H2O
if omitted
Template total RNA
(5ng/μL)
Total volume
0.5
1
2
4
10
20
Table 21. Reverse transcription reaction setup per sample1)
The amount of 100x fold diluted cDNA needed for the different
Pick-&-Mix layouts can be seen in Table 22 – Step 7.
Mix the reaction by very gentle vortexing or pipetting to ensure that all
reagents are thoroughly mixed. After mixing, spin down.
1) The amount of cDNA required per sample depends on the number of microRNAs analyzed per sample. The volumes suggested here
provides excess amount of cDNA, which ensures the highest possible reproducibility because these volumes can be pipetted with
great accuracy.
47
Protocol D. Pick-&-Mix
microRNA PCR Panels
Step 4
Mix and spin reagents
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 5
Incubate and
heat inactivate2)
• Incubate for 60 min at 42˚C.
• Heat-inactivate the reverse transcriptase
for 5 min at 95˚C.
• Immediately cool to 4°C.
• Store at 4°C or freeze.
qPCR protocol:
Step 6
Prepare reagents
for real-time PCR
Place cDNA (from Step 5), nuclease free water and PCR Master mix
on ice and thaw for 15-20 min. Protect the PCR Master mix vials from
light. Immediately before use, mix the PCR Master mix by pipetting up
and down. The rest of the reagents are mixed by vortexing and spun
down.
Protocol D. Pick-&-Mix
microRNA PCR Panels
2) Although not recommended, the protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks
(optional store at 4˚C for up to 4 days). It is recommended that synthesized cDNA be stored in “low-nucleic acid binding” tubes or plates.
48
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 7
Dilute cDNA template
100x in nuclease
free water3)
Dilute the cDNA from the RT reactions to give a final 100x dilution.
Suggested cDNA dilution procedures is shown in Table 22 along with
required volumes of diluted cDNA for the different Pick-&-Mix predefined layouts. It is receommeded that “low-nucleic acid binding”.
For fully customized layouts dilutions may be adjusted to the specific
replicate scheme. tubes or plates are used. It is not recommended to
store the 1:100 dilution of cDNA.
Pick-&-Mix plate
configuration
8x10 (12)
Suggested
cDNA dilution
procedure
cDNA + nuclease
free water (μL)
2 + 198
Volume (μL) of
diluted cDNA
needed for each
Pick-&-Mix plate
60
4x22 (24)
2 + 198
120
1x92 (96)
5 + 495
480
16x22 (24)
2 + 198
120
8x46 (48)
3 + 297
240
4x94 (96)
5 + 495
480
1x380 (384)
20 + 1980
1920
Table 22. Amount of 100x diluted cDNA needed in
Pick-&-Mix plates
Recommendation: Include a passive reference dye in the cDNA
dilution if advised by instrument manufacturer. Please note that the
PCR Master mix does not include ROX. The amount of ROX required is
instrument dependent and it is important to refer to the manufacturer’s
recommendations when deciding how much ROX to use, see Tip 8.
See tip 8
3) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.
49
Protocol D. Pick-&-Mix
microRNA PCR Panels
Numbers in parenthesis designate total number of assays/sample,
including controls and inter-plate calibrators. Loss from pipetting is
not included in the volumes. listed in the right column (diluted cDNA
needed).
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
ABI instrument user?
Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file
Step 8
Combine cDNA and
PCR Master mix 1:1
and add to PCR plates
Mix thoroughly
The following procedure is recommended:
1. Before removing the plate seal, briefly spin down the plate(s) in a
plate centrifuge.
2. Combine 2x PCR Master mix and 100x diluted cDNA 1:1 (e.g. 500 μL
2x PCR master mix and 500 μL diluted cDNA).
3. Mix gently by inverting the tube, spin down.
4. Add 10 μL PCR Master mix:
cDNA mix to each well.4)
5. Seal the plate with optical sealing as recommended by the
instrument manufacturer.
The experiment can be paused at this point. Store the reactions
protected from light at 4°C for up to 24 hours.
Step 9
Spin plate
Spin plate briefly in a plate centrifuge (1500g for 1 minute), to remove
air bubbles.
Step 10
Real-time
PCR amplification
Perform real-time PCR amplification followed by melting curve
analysis according to Table 23.
Protocol D. Pick-&-Mix
microRNA PCR Panels
Process step
Polymerase Activation/
Denaturation
Amplification
Melting curve analysis8)
Settings, LC480 Settings, other
instrument5)
instruments6)
95˚C, 10 min
95˚C, 10 min
45 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s7)
Optical read
Yes
Table 23. Real-time PCR cycle conditions
50
40 amplification
cycles at
95˚C, 10 s
60˚C, 1 min,
ramp-rate
1.6˚C/s7)
Optical read
Yes
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Step 11
Analyze data
Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on
PCR instrument). If you are using an ABI instrument, please note that
it is not recommended to use auto Ct settings. For a guide on how to
set manual baseline and threshold, refer to Tip 10, page 56 in the tips
section. Furthermore, if the data is to be analyzed using Exiqon GenEx,
the experiment must be set up as an AQ experiment, not RQ.
For tips on normalization, please see Tip 11, page 58. If you are using
a Roche LC480 instrument, we recommend analysis using the 2nd
derivative method. We recommend performing normalization and
further data analysis with the Exiqon GenEx qPCR analysis software
(www.exiqon.com/mirna-pcr-analysis). Please refer to our data
analysis guide for recommendations.
See tip 11
4) Corresponding to 0.05ng total RNA starting material pr. PCR reaction.
5) Five additional amplification cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40.
6) If using a 96-well cycler with a minimum recommended volume of 20 μL (like some ABI instruments), then use 10 μL reaction volume
and set the instrument settings at 20 μL.
7) The ramp-rate of cooling from 95˚C to 60˚C should be set to 1.6°C/s. This is equivalent to 100% under standard cycling conditions on
the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised.
8) Melting curve analysis of the PCR product(s) is recommended to verify specificity and identity of the amplification reaction. Melting
curve analysis is an analysis step built into the soware of instruments. Please follow the instructions provided by the supplier.
Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
Protocol D. Pick-&-Mix
microRNA PCR Panels
51
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Tips to protocol
Tip 1. RNA extraction and template preparation
Purification and preparation of total RNA that includes small RNAs (<200 nt) from a
biological sample is the first critical step for a successful expression profiling analysis
of microRNAs. Therefore, the method used for RNA sample preparation is critical to the
success of the experiment.
The following points should be considered before starting the experiment:
• We recommend using the miRCURY™ RNA Isolation kit for isolation of RNA that contains
the small RNA fraction. If not using one of the miRCURY™ RNA Isolation kits, it is
important that the method used to isolate RNA from a sample should give a quantitative
recovery of small RNAs and should not result in a substantial loss of the small RNA
fraction. This also applies when using commercially available kits. Make sure the total
RNA preparation is guaranteed to contain microRNA.
• If commercially available purified RNA is used, it is important to make sure that the
RNA is guaranteed to contain small RNAs and is representative of the microRNAs in the
species and/or tissue from which it was isolated.
• The comparison of samples prepared using different RNA isolation methods is not
recommended. However, if this is necessary, it is recommended to include the measure
of a reference small RNA which has a consistent and unvaried expression level in order to
allow for comparison of microRNA levels in the different sample preparations.
• The isolation of RNA and the reaction steps preceding real-time PCR should be performed
in rooms separate from where real-time PCR experiments will take place in order to avoid
contamination with amplicon.
• It is recommended that the integrity of isolated RNA be assessed before proceeding with
quantitative real-time PCR experiments. This may be performed either on the Agilent
2100 Bioanalyzer or by denaturing gel electrophoresis.
• If necessary, treat RNA preparations with DNases to remove contaminating DNA that may
interfere with the real-time PCR results.
• Purified total RNA should be dissolved in nuclease-free water at a stock concentration of
at least 1 μg/μL. See recommendations for storage conditions in Tip 3.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Tip 2. Guidelines for serum, plasma and biofluid samples
Plasma and serum are essentially cell free liquid samples. This means that only circulating
RNA is extracted from these sample types, resulting in low total RNA concentrations,
even if the microRNA fraction is readily detectable. The result of this is that measuring
correct RNA concentrations is difficult, and that there is a high risk of increased loss during
extraction. For this reason, we recommend using RNA amounts based on starting volume
rather than RNA quantity. Comprehensive guidelines for microRNA profiling in blood serum/
plasma can be downloaded at www.exiqon.com/serum-plasma-guidelines.
Tip 3. General guidelines for handling and storage of RNA
The following precautions should be taken to prevent RNase contamination and degradation
of the RNA sample and reagents:
• Always wear disposable gloves.
• Use nuclease-free, low nucleic acid binding plasticware and filter barrier pipette tips.
• Keep tubes capped when possible. Always spin tubes before opening.
• For long-time storage, RNA may be stored at -80°C. Avoid repeated freezing and thawing
cycles.
Tip 4. Good PCR laboratory practice
To reduce the risk of contaminating PCRs with “old” PCR amplicons, and consequently
obtain false results:
• Always wear a clean lab coat. Use separate lab coats for RNA sample preparation, cDNA
synthesis and when setting up PCR reactions or handling PCR products.
• Change gloves oen, especially whenever you suspect they may have been contaminated.
• Establish and maintain designated areas for PCR set-up, PCR amplification, and gel
electrophoresis of PCR products.
• Never bring amplified PCR products into the PCR set-up area.
• Spin down all reaction and sample tubes before opening. Open and close all reagent and
sample tubes carefully, trying not to splash or spray PCR samples.
• Keep reactions and components capped whenever possible.
• Use filter barrier pipette tips to avoid aerosol-mediated contamination of your pipetting
device.
• Clean laboratory benches and equipment regularly.
53
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Tip 5. Keep reagents and reactions cool at all times
In order to ensure optimal performance of the miRCURY LNA™ Universal RT microRNA
PCR system it is important that the reagents and reactions are kept on ice (or at 4°C) as
much as possible during the protocol (apart from reaction steps specifically involving raised
temperatures).
Tip 6. First strand cDNA synthesis
Store cDNA samples in nuclease-free low nucleic acid-binding micro centrifuge tubes, e.g.
Eppendorf DNA LoBind tubes.
Tip 7. Recommended controls
The following controls are recommended and should be included in the experimental set-up:
• Reverse transcription/no enzyme controls, i.e. first-strand cDNA synthesis reactions
performed without the Enzyme mix. If a PCR product is amplified from this control
reaction it indicates genomic DNA or PCR product contamination of the template RNA.
• Non-template controls in the real-time PCR amplification, i.e. real-time PCR
reactions performed without any cDNA template. This control will reveal PCR product
contamination of the reaction.
• Blank purification or carrier only, i.e. when purifying RNA in the presence of carrier RNA
(such as for serum and plasma samples). This control will reveal any non-specific signals
originating from the carrier RNA alone.
Tip 8. Passive reference dye (ROX)
Many real-time PCR instruments will only produce reliable results when a passive
reference dye such as ROX is added to the PCR reaction. The reference dye is used to
normalize signals from individual PCR wells in order to enable comparison of real-time PCR
amplification signals across an entire PCR-plate.
It is recommended to determine whether your real-time PCR instrument has this type
of requirement. The amount of ROX to include in the PCR reaction depends on the
requirements of the real-time PCR instrument and must be adjusted accordingly. Please
follow the supplier’s instructions for preparation and concentrations of ROX solutions.
Typically, real-time PCR instruments that allow excitation at individual wavelengths for
54
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
individual dyes (most filter wheel based instruments) require less ROX than instruments that
use a single excitation wavelength for all fluorophores (most laser based instruments use
excitation at 488 nm). It is important to note that excessive amounts of ROX may inhibit the PCR
reaction. It may be recommended to perform a titration of ROX amounts in order to determine
the optimal concentration for a particular instrument-system combination. It is possible to use
the spike-in template (provided in the Universal cDNA synthesis kit II) and the Control primers
(provided in the ExiLENT SYBR® Green master mix kit) to perform such titrations.
Recommended ROX concentrations are found here:
Instrument
ROX concentration
ABI 7900HT
300-500 nM
ABI 7900HT FAST
300-500 nM
ABI Viia7
30-50 nM
ABI Viia7 FAST
30-50 nM
ABI 7500 FAST
30-50 nM
ABI StepOnePlus
300-500 nM
ABI 7300
300-500 nM
ABI 7500
30-50 nM
ABI 7700
300-500 nM
ABI 7000
300-500 nM
Table 24.
Tip 9. Inter-plate calibration
When setting up large scale experiments with several PCR plates involved, individual run
differences may be observed. One way of avoiding these having an effect on the results
is setting up the experiment in such a way that all samples for one gene are run on the
same plate. However, this is not always feasible. When Cq values for one gene have to be
compared across plates, it is recommended to employ an inter-plate calibrator.
An inter-plate calibrator is a template-assay combination which is the same in all plates,
and always located in the same well across different plates. This can then be used to
calibrate all plates to give the same Cq value for the calibrator, thereby reducing run-to-run
variance. Please note that all panels contain inter-plate calibrators.
55
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Tip 10. Guidelines for real-time PCR data collection using ABI instruments
On cyclers using baseline and threshold values for Cq (Ct) calculations, such as ABI 7900HT,
it is important that the proper settings are used. Use of the automatic function of the
soware for these settings does not seem to produce optimal results for SYBR® Green
based assays. Oen the baseline is set erroneously on non-detected assays, and this in turn
gives false positives, therefore do not use automatic settings. Another issue to consider
when using automatic settings is that the settings may differ between plates resulting in
data that cannot be compared directly. Inter-plate calibration may not fully resolve this
issue, since each assay has a separately calculated baseline and threshold. Instead, both
threshold and baseline should be set manually, applying the same settings for all assays on
the plate.
The following principles should be applied to manual baseline and threshold settings:
Baseline:
The baseline should be calculated in the cycle interval before the amplification takes off
(see Figure 3).
Threshold:
The threshold should then be set with the Y-axis in log scale where all assays are in the log
linear phase, and the threshold above background for all assays (see Figure 3).
Note: The optimal threshold value may vary between individual machines and experiments.
Important note
If ROX passive reference dye has not been used in the PCR reactions, make sure the
SDS soware is set-up without reference dye correction.
56
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Baseline setting
Threshold above
background
Amplification
take-off
Baseline
interval
Figure 5.
57
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Tip 11. Quick guide to normalization of microRNA qPCR experiments
The purpose of normalization is to remove technical and biological variation between
samples that is not related to the biological changes under investigation. Proper
normalization is critical for the correct analysis and interpretation of results from realtime PCR experiments. The most commonly used option for normalization is to use stably
expressed reference genes.
In general it is recommended to test several endogenous control candidates (reference
genes) before setting up the actual microRNA expression analysis. These candidates should
be chosen among genes that can be expected to be stably expressed over the whole range
of samples being investigated. They can be stably expressed small non-coding RNA, or
stably expressed microRNAs, and chosen based on literature or pre-existing data (e.g.
microarray analysis or qPCR panel screening).
Exiqon offers primer sets for a number of different small RNAs which tend to be stably
expressed, and are therefore oen good candidates for reference genes. It’s important to
keep in mind that in spite of being small non-coding RNAs, most of these are significantly
larger than microRNA and therefore may have different extraction efficiency and stability.
U6 is one such reference gene which is oen used. However, U6 is significantly larger than
microRNAs and has a different sub-cellular distribution. The existence of several different
isoforms also makes it a suboptimal reference gene. 5S ribosomal RNA is another popular
option, but this RNA has a much higher expression level than most microRNAs, and is oen
found as a PCR contaminant.
If working with blood serum or plasma, please note that only circulating RNA are present.
In this case the small non-coding RNAs (5S, U6, SNORs etc) are not good candidates for
reference genes, since they are most probably not present in the sample.
58
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Using stably expressed microRNAs as reference genes offers several advantages such as
equal size, extraction efficiency and stability, as well as having expression levels within a
similar range of the target microRNAs. Several candidates can be found in the literature,
including miR-191-5p, miR-103a-3p, let-7a-5p, and miR-16-5p. Microarray or qPCR panel
screening data may also be used when choosing candidate reference genes.
All reference gene candidates should be empirically validated for each study. A number
of different soware packages exist for evaluating the optimal nature and number of
endogenous controls, and for applying multiple endogenous controls for normalizing
target expression. One such option is the GenEx soware from MulitD, sold through Exiqon
with a special application for Exiqon PCR panels. GenEx incorporates both GeNorm and
Normfinder for finding the optimal reference genes, and is easy and intuitive to use for the
actual normalization.
An alternative option for normalization of data from panels (profiling a high number of
microRNAs) is to normalize against the global mean; that is, the average of all expressed
microRNAs This can be a good option in samples with a high call-rate (expressed
microRNAs), but should be used with caution in samples with low call-rates. It is also not a
good option in samples for which the general microRNA expression level is changed.
For further information on normalization and references, we recommend our data-analysis
guide available online as well as the guide to microRNA normalization from
www.gene-quantification.de.
Tip 12. Melting curves
Melting curves are a good tool for evaluating the specificity of the assay. When setting up
the melt curve temperature gradient on the instrument, it is recommended to start at 60˚C.
59
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Troubleshooting guide
Problem
Suggestion
PCR signal in samples
amplified from firststrand synthesis
reactions performed
without reverse
transcriptase
This typically indicates contamination of the template RNA with
genomic DNA. Perform DNase treatment of the RNA sample. If this
does not solve the problem RNA samples or other reagent may be
contaminated with PCR products.
PCR signal in
no-template
PCR reaction
This typically indicates contamination of the cDNA template or PCR
reagents with amplified PCR product.
Exposing the reactions to elevated temperatures (i.e. room
temperature) during any part of the protocol increases the risk of
background signals. It is important that the reagents and assembled
reactions are kept cool (on ice or 4°C) at all times (see Tip 4,
page 53 for details).
Generated signals
are weak
• On some real-time PCR cyclers, gain-settings are adjustable.
Make sure the gain settings of your real-time PCR cycler have been
set to accommodate the signals generated from the specific assay.
• RNA samples may contain PCR inhibitors. Further purification or
an alternative RNA extraction method may be necessary. Check
positive controls.
No fluorescent signal is
detected during the PCR
Confirm that you have a PCR product by running an aliquot of your
PCR reaction on an agarose gel.
No fluorescent signal
detected during the PCR,
but a PCR amplicon can
be detected by agarose
gel electrophoresis
• Check that the filter in the real-time PCR cycler was set to either
SYBR® Green or FAM/FITC.
• Check that the optical read is at the correct step of the real-time
PCR cycles.
• Adjust the baseline in the real-time PCR cycler software.
60
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
FAQs
What kind of real-time PCR instruments is miRCURY LNA™ Universal RT microRNA
PCR compatible with?
miRCURY LNA™ Universal RT microRNA PCR is compatible with all instruments capable of
reading green fluorophores such as fluorescein/FITC/FAM and SYBR® Green. The system has
been tested and found to work on real-time PCR instruments from several leading suppliers
of this type of instrument.
What kind of settings should I use on my real-time PCR instrument?
If your real-time PCR instrument supports fluorophores such as fluorescein/FITC/FAM or
SYBR® Green your instrument must be set to detect these fluorophores.
Is miRCURY LNA™ Universal RT microRNA PCR compatible with other SYBR® Green
master mixes?
We do not recommend using other SYBR® Green master mixes for real-time PCR analysis
with the LNA™ PCR primer sets or Ready-to-Use panels. The primer sets have been
optimized and validated using the miRCURY LNA™ ExiLENT SYBR® Green master mix and
the performance of the primer sets will be compromised by using a different master mix
(which may contain different salt and/or enzyme concentrations).
My RNA is already enriched for microRNA, how much should I use in the real-time PCR
experiments?
miRCURY LNA™ Universal RT microRNA PCR is developed for use on total RNA and we do
not recommend enriching for small RNAs. Samples of enriched microRNAs are difficult to
quantitate accurately making it very tricky to ensure the same amount of sample is added
to each reaction. If necessary, a total RNA equivalent should be used for the enriched
sample, e.g. use a proportional amount of enriched sample resulting from 20 ng of total
RNA. It may be necessary to try a couple of different amounts of enriched sample to ensure
that the results fall within the linear range of the assay.
61
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
What is the recommended experimental set up for Universal RT microRNA real-time PCR?
It is generally accepted that the reverse transcription (RT) reaction gives rise to more
variation than the PCR reaction. It is therefore advisable to perform replicate RT reactions,
ideally 3 separate reactions with 1-2 PCR reactions for each RT. It is further recommended
to always include at least three biological replicates (separate RNA extractions) of
each sample type in order to allow statistical analysis of the results. If small changes in
microRNA expression are expected, it may be necessary to include more replicates to
ensure a signifi cant result. In general it is recommended that replicates should be included
at any stage during sample procurement, processing, RNA isolation, etc. that could give rise
to variation between samples.
A tech note on guidelines for setting up microRNA qPCR experiments can be downloaded at
www.exiqon.com/miRNA-qPCR-guidelines
Additional information is available at www.exiqon.com/mirna-pcr
62
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Related products
Exiqon offers a broad variety of tools enabling new discoveries concerning the expression,
function and spatial distribution of microRNAs:
miRCURY LNA™ microRNA Array, microarray kit
Pre-printed miRCURY LNA™ microRNA Array microarray slides, available for hsa, mmu
& rno and other species. Kit includes hybridization and wash buffers as well as synthetic
spike-in microRNAs.
miRCURY LNA™ microRNA Power labeling kit
For fluorescent labeling of microRNAs from total RNA samples ready for array
hybridization.
miRCURY LNA™ microRNA Array, ready-to-spot probe set
Ready-to-spot oligos for direct printing of arrays, or coupling in bead-based applications.
miRCURY LNA™ microRNA Detection Probes
For in situ hybridization and northern blotting of all annotated microRNAs.
miRCURY LNA™ microRNA ISH Optimization kit (FFPE)
Complete kit with control probes and hybridization buffer for easy set up of microRNA
in situ hybridization.
miRCURY LNA™ microRNA Inhibitors, Power Inhibitors and Family Inhibitors
Unravel the function of microRNAs by microRNA inhibition. Sophisticated LNA™ design
ensures potent inhibition of all microRNAs regardless of their GC content. Chemically
modified, highly stable Power Inhibitors for unrivalled potency. Available for in vitro and
in vivo studies.
miRCURY LNA™ microRNA Inhibitor Library
For genome-wide high throughput screening of microRNA function.
miRCURY LNA™ Target Site Blockers
For inhibition of microRNA binding to a specific mRNA target.
63
EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Notice to purchaser
Exiqon, LNA and miRCURY are registered trademarks of Exiqon A/S, Vedbaek, Denmark. SYBR Green is a licensed
trademark of Invitrogen. All other trademarks are the property of their respective owners.
Locked-nucleic Acids (LNAs™) are protected by US Pat No. 6,268,490, US Pat No. 6,770,748, US Pat No. 6,639,059,
US Pat No. 6,734,291 and other applications and patents owned or licensed by Exiqon A/S. Products are provided
to buyers for research use only. The products in their original or any modified form may be used only for the buyer’s
internal research purposes and not for commercial, diagnostic, therapeutic, or other use, including contract research.
The buyer may not provide products to third parties in their original or any modified form. The purchase of products
does not include or carry an implied right or license for the buyer to use such products in their original or any modified
form in the provision of services to third parties, and a license must be obtained directly from Exiqon A/S for such
uses.
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside
the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, non transferable immunity from
suit under the foregoing patent claims for using only this amount of product solely in Contract Research, including
reporting results of purchaser’s activities for a fee or other commercial consideration, and also for the purchaser’s
own internal research. No right under any other patent claim is conveyed expressly, by implication, or by estoppel.
Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Furthermore, this product is provided under an agreement between Molecular Probes, Inc., a wholly owned subsidiary
of Invitrogen Corporation, and EXIQON and the manufacture, use, sale or import of this product is subject to one or
more U.S. Patents and corresponding international equivalents. The purchase of this product conveys to the buyer
the non-transferable right to use the purchased amount of the product and components of the product in research
conducted by the buyer, where such research does not include testing, analysis or screening services for any third
party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its
components or (c) materials made using this product or its components to a third party or otherwise use this product
or its components or materials made using this product or its components for Commercial Purposes. Commercial
Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product
or its components in manufacturing; (2) use of the product or its components to provide a service, information, or
data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of
the product or its components, whether or not such product or its components are resold for use in research. For
information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc.,
Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300, Fax: (541) 335-0354.
Further, the purchase of this product includes a limited, non-transferable license under specific claims of U.S.
Patent Nos. 6,174,670 and 6,569,627, owned by the University of Utah Research Foundation and licensed to Roche
Diagnostics GmbH and Idaho Technology, Inc., to use only the enclosed amount of product according to the specified
protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any
claim of U.S. Patent Nos. 6,174,670 and 6,569,627, other than for the amount of product contained herein.
For life science research use only. Not for use in diagnostic procedures.
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EXIQON | miRCURY LNA™ Universal RT microRNA PCR | Instruction manual
Notes
65
North America
Exiqon Inc.
12 Gill Street, Suite 1650
Woburn, MA 01801, United States
Phone (781) 376 4150
Fax (781) 376 4152
exiqon.com
13-0099 - 920003 - v6.2 - 07/2016 - NORMAL
Outside North America
Exiqon A/S
Skelstedet 16
DK-2950 Vedbaek, Denmark
Phone +45 45 650 929
Fax +45 45 661 888