High Pure miRNA Isolation Kit
Flexible, organic-solvent-free isolation of
small RNA
Choose from the High Pure miRNA Isolation Kit’s one- or
two-column protocols to easily isolate either high-purity total
RNA that includes miRNA, or enriched miRNA (Figure 1).
Eliminate the need for hazardous organic
solvents.
Avoid organic solvents while isolating RNA from a variety of
sample materials: mammalian cells and tissue, plant tissue,
and formalin-fixed, paraffin-embedded (FFPE) tissue samples.
Obtain high yields with a simple, efficient
protocol.
Use the purified RNA directly in miRNA array hybridization,
northern blotting, or relative quantification of miRNA by
real-time PCR.
Experts at Extraction
One of the few mammals to use tools, the Sea Otter (Enhydra lutris) uses
rocks or other objects to crack open shellfish to extract its meal.
Isolate high-quality RNAs without using toxic phenol/
chloroform.
Recover high yields of purified total RNA and miRNA
through a straightforward protocol (Figures 2 and 4).
Choose one flexible kit for all your miRNA
purifications.
Use the same versatile kit to purify small RNAs from a variety
of sample types, including both fresh and FFPE tissues
(Figure 3).
Eluate 1
Total RNA
Eluate 2
Eluate 3
Purified small RNA
5.8S RNA
t-RNA
miRNA 145
(spiked in)
1 column
2 column
Figure 1: RNA isolated using the one- and two-column protocols of the
High Pure miRNA Isolation Kit.
Eluate 1: N
ucleic acids isolated with the one-column protocol (total RNA
including miRNA).
Eluate 2: N
ucleic acids bound to the first column in the two-column protocol.
Eluate 3: E
nriched miRNA recovered using the two-column miRNA isolation
protocol.
Results: The High Pure miRNA Isolation Kit efficiently isolates total RNA
containing miRNA, and enriches miRNA by removing large RNA fragments.
www.roche-applied-science.com
Efficiently isolate high-purity RNA for direct use in downstream applications
One-column protocol
Two-column protocol
150 µl lysate supernatant
add 312 µl Binding Buffer +
200 µl Binding Enhancer
150 µl lysate supernatant
Mix well and apply mixture
(max. 700 µl at a time) to a
High Pure filter tube
assembly, centrifuge at
13,000 × g for 30 - 60 s
(repeat if lysate volume
is more than 700 µl).
add 312 µl Binding Buffer
Mix well and apply mixture
(max. 700 µl at a time) to a
High Pure filter tube
assembly, centrifuge at
13,000 × g for 30 – 60 s
(repeat if lysate volume
is more than 700 µl).
Add 5 00 µl Wash Buffer
Disc ard flowthrough
Centrifuge at 13,000 × g
for 30 s
collect flowthrough
Add 200 µl Binding
Enhancer
Add 3 00 µl Wash Buffer
Disc ard flowthrough
Centrifuge at 13,000 × g
for 30 s
Mix well and apply mixture
to a new High Pure filter
tube assembly, centrifuge
at 13,000 × g for 30 s.
Disc ard flowthrough
Centrifuge at 13,000 × g
for 1 min
Add 500 µl Wash Buffer
Centrifuge at 13,000 × g
for 30 s
Discard collection tube
Place the Filter Tube
in a fresh1.5 ml
microcentrifuge tube
Add 300 µl Wash Buffer
Disc ard flowthrough
Centrifuge at 13,000 × g
for 30 s
Add 50 - 100 µl Elution
Buffer on the center
of the filter
Disc ard flowthrough
Incubate for 1 min
at +15 to +25°C.
Centrifuge at 13,000 × g
for 1 min
Centrifuge at 13,000 × g
for 1 min
Discard collection tube
Place the Filter Tube in a
fresh 1.5 ml
microcentrifuge tube
Purified total RNA
Figure 2: Choose the High Pure miRNA isolation Kit one-column
protocol to isolate total RNA including the small RNA fraction.
Add 50 - 100 µl Elution
Buffer on the center
of the filter
Incubate for 1 min at
+15 to +25°C.
Centrifuge at 13,000 × g
for 1 min
60
Purified small RNA
Figure 4: Use the High Pure miRNA Isolation Kit two-column protocol to
isolate enriched miRNA.
40
In the first step, large RNA molecules are bound to the silica matrix of the first
column and thus removed from the sample. The flowthrough is then combined
with Binding Enhancer (provided in the kit) and placed on the second spin
column to isolate the enriched miRNA fraction of the sample.
µg RNA
50
30
20
Ordering information
10
0
Heart
Liver
Muscle Pancreas Kidney
Brain
Lung
Intestine
Blue: FFPE tissue / Supplier 1 Kit
Red: FFPE tissue / Supplier 2 Kit
Yellow: FFPE tissue / High Pure miRNA Isolation Kit
Green: Fresh-frozen tissue extracted with phenol/chloroform
Figure 3: Yield comparison of RNA isolated from fresh-frozen or FFPE
mouse tissues using phenol/chloroform extraction or kits from various
manufacturers.
Half of each tissue sample was fresh-frozen, and the other half was formalinfixed and paraffin-embedded (FFPE). The fresh-frozen samples were extracted
with a phenol/chloroform reagent to isolate RNA. Equal amounts (10 µm
sections) of FFPE samples were used for RNA isolation with either the High
Pure miRNA Isolation Kit or kits from other suppliers following manufacturers’
instructions. Isolated total RNA from each preparation was quantified on a
NanoDrop instrument.
Results: The High Pure miRNA Isolation Kit shows an overall superior yield
compared with kits from two other suppliers, and no significant RNA loss
compared with a phenol/chloroform-based isolation method (data provided by
Exiqon A/S).
Product
Cat. No.
Pack Size
High Pure miRNA Isolation Kit
05 080 576 001
Up to 50 isolations
For more information about the High Pure miRNA Isolation
Kit and other products for nucleic acid isolation and purification, visit www.roche-applied-science.com/napure
HIGH PURE is a trademark of Roche.
Other brands or product names are trademarks of their respective holders.
Published by
Roche Diagnostics GmbH
Roche Applied Science
68298 Mannheim
Germany
©2008 Roche Diagnostics GmbH
All rights reserved.
052049250010308