I N T EG R AT E D DNA
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triFECTin
TM
TRANSFECTION REAGENT
ABSTRACT
TriFECTinTM - Cationic Lipid-Based Transfection of Mammalian Cells
Ashley Jacobi, Kim Lennox, Michael Collingwood,
Scott Rose Ph.D., & Mark Behlke M.D., Ph.D.
The introduction of short single or double-stranded oligonucleotides (RNA or DNA) into primary or immortalized cell lines
is usually accomplished through use of cationic lipids or mixtures of cationic lipids and cationic polymers as transfection
reagents. Dozens of different lipid-based transfection reagents are on the market today, each of which has certain
benefits and drawbacks. Unfortunately, no single reagent or transfection method can be universally used to transfect
all kinds of nucleic acids into all cell types. There is often a trade-off between efficient delivery and toxicity. Different
cell types often require use of different reagents, and some cells seem to be almost totally resistant to lipid-mediated
transfection. As an added complication, different types of nucleic acids also may require use of different reagents; the
optimal transfection protocol for a large double-stranded DNA plasmid is likely to be different for a short single-strand
antisense oligo (ASO) or dsRNA, such as an siRNA. Chemical modifications, such as phosphorothioate bonds or 2’-alkyl
sugars (2’-O-Methyl RNA) may also alter the transfection behavior of a nucleic acid 1. Sophisticated labs often keep a
half dozen or more different cationic lipids on hand to optimize transfection for various types of nucleic acids in use in
different cell lines.
To help solve some of these problems, IDT offers Trifectin, a transfection reagent that is very effective at delivering most
types of synthetic oligonucleotides into cells in culture with minimal toxicity. In particular, the Trifectin formulation is
optimized to deliver our Dicer-substrate siRNAs (double-stranded RNAs) and anti-miRNA antisense oligos (single-stranded
phosphorothioate chimeras containing DNA, LNA, and/or 2’-O-Methyl RNA bases). Trifectin is amazingly non-toxic, and as
much as 10x the optimal concentration can be used without inducing cell death.
Elements of successful RNAi-directed gene silencing or miRNA knockdown experiments include:
1. a potent siRNA or ASO.
2. an effective non-toxic transfection reagent that directs entry of the nucleic acid into the cell with entry into the
right intracellular compartments.
3. a good qPCR assay to rapidly assess efficacy of RNA knockdown.
©2008 Integrated DNA Technologies
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I N T EG R AT E D DNA
T E C H N O LO G I E S
TECHNICAL
REPORT
TriFECTinTM - Cationic Lipid-Based Transfection of Mammalian Cells
Ashley Jacobi, Kim Lennox, Michael Collingwood,
Scott Rose Ph.D., & Mark Behlke M.D., Ph.D.
Potent Knockdown Reagents - DsiRNAs
IDT offers predesigned Dicer-substrate siRNAs (DsiRNAs) against all genes in the human, mouse, rat, cow, dog, chicken,
and chimp transcriptomes. These DsiRNAs have been shown to be very potent both in vitro and in vivo2-4. Validated Dicersubstrate siRNAs are available from BioRad (siLentMersTM).
Transfection Reagent Validation
For an RNAi or antisense experiment, the actual level of target gene knockdown directly relates to the transfection efficiency.
If a DsiRNA has a theoretical 90% knockdown capability and transfection is 75% efficient then an actual knockdown of 0.9 x
0.75 = 0.675 (67.5%) will be seen in the real experiment. Considering how important transfection is to the end results of your
experiment, it is prudent to optimize transfection conditions for each type of nucleic acid and each cell type used.
Typically IDT optimizes transfection and validates functional potency of new reagents in a 3-step process:
1. Dye-labeled oligo transfection
a. This permits rapid testing of a variety of reagents. Just transfect, wash the cells, and look for fluorescence.
b. If the cells do not show uptake of the fluorescent reagent, then this combination of lipid + nucleic acid class + cell
type probably is not worth pursuing. It is easy to test multiple lipid/RNA complexation ratios, etc. in this manner at
low cost and with minimal effort.
c. However, fluorescence does not necessarily mean that the dye-labeled oligo has been delivered to the right
intracellular location; therefore move on to step 2. A dye-labeled oligo sequestered in an endosome can fluoresce
but may still not be available to enter RISC and trigger RNAi.
2. Positive control transfection
a. Test real functional delivery. Is the DsiRNA or ASO introduced into the correct intracellular compartments to show
activity?
b. Establish the functional efficiency of transfection. If a positive control known to give 95% knockdown in one cell line
only gives 80% knockdown in another cell line, then more optimization may be needed before moving forward.
c. IDT has a full line of DsiRNA control products, including validated anti-HPRT control DsiRNAs for use in human,
mouse, rat, Chinese hamster, cow, and pig cells. PCR primers suitable for SYBRTM Green qRT-PCR assays are available
as well as cloned amplicons (plasmids) to allow absolute quantification with standard curves.
d. Validated DsiRNAs are also available for reporter constructs, such as EGFP, FLuc (pGL2,3), FLuc (pGL4), and RLuc.
3. Functional testing of new reagents
a. Once an efficient transfection protocol has been established, then new reagents can be characterized.
b. We recommend performing dose response testing to establish the real potency of new reagents before use in functional experiments.
Use of Trifectin for RNAi Applications
Figure 1 shows results from a typical transfection validation experiment. Trifectin was employed with the human glioblastoma cell line T98G (ATCCTM CRL-1690TM). Fluorescent uptake of a dye-labeled RNA duplex is shown (TEXTM615 DS Transfection
Control) along with functional knockdown of the HPRT-1 gene (hypoxanthine phosphoribosyltransferase 1) using the HPRT
positive control DsiRNA.
©2008 Integrated DNA Technologies
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I N T EG R AT E D DNA
T E C H N O LO G I E S
TECHNICAL
REPORT
Figure 1a
Phase Contrast
Fluorescence
Figure 1
T98G Cells transfected with TriFECT in
Reduction of HPRT-1 mRNA levels using Trifectin in T98G Cells
% HPRT mRNA Levels
T98G cells were reverse transfected with 10 nM of a TEXTM615-dye
labeled RNA duplex or 10 nM, 1 nM, or 0.1 nM of HPRT or Negative
Control DsiRNAs, using 2 µL of Trifectin in a volume of 600 µL. Cells
were transfected at a density of 6x104 cells per well in a 24-well
plate in complete media minus antibiotics. The transfection mixture
was left on the cells for 24 hours. Cells were washed before imaging. 1a. Fluorescent cell images were obtained using an Olympus
1X51fluorescent microscope using a 40X objective. 1b. RNA was isolated and qRT-PCR was performed to determine HPRT mRNA levels.
HPRT values were normalized to the RPLP0 gene (internal control)
and set for the Neg Con = 100%.
120
10 nM
1 nM
100
0.1 nM
80
60
40
20
0
Neg Con
HPRT
DsiRNA Duplex
Figure 1b
Figure 2a
Phase Contrast
Fluorescence
Figure 2
HepG2 Cells transfected with TriFECT in
Reduction of HPRT-1 mRNA levels using Trifectin in HepG2 Cells
% HPRT mRNA Levels
HepG2 cells were reverse transfected with 10 nM of a TEXTM615-dye
labeled RNA duplex or 10 nM, 1 nM, or 0.1 nM of HPRT or Negative
Control DsiRNAs, using 4 µL of Trifectin in a volume of 600 µL. Cells
were transfected at a density of 1.1x105 cells per well in a 24-well
plate in complete media minus antibiotics. The transfection mixture
was left on the cells for 24 hours. Cells were washed before imaging. 1a. Fluorescent cell images were obtained using an Olympus
1X51fluorescent microscope using a 40X objective. 1b. RNA was
isolated and qRT-PCR was performed to determine HPRT mRNA levels.
HPRT values were normalized to the RPLP0 gene (internal control) and
set for the Neg Con = 100%.
120
10 nM
1 nM
100
0.1 nM
80
60
40
20
0
Neg Con
HPRT
DsiRNA Duplex
Figure 2b
©2008 Integrated DNA Technologies
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I N T EG R AT E D DNA
T E C H N O LO G I E S
TECHNICAL
REPORT
Use of Trifectin for Antisense and Anti-miRNA Applications
Introducing modified antisense DNA oligonucleotides (ASO) into cells can be difficult, especially if the oligonucleotide is
extensively modified. Historically, LipofectinTM (or related formulations) has been used to deliver phosphorothioate-modified
oligos, but unfortunately these reagents can be toxic to many cell types. Trifectin is as effective with heavily modified oligos
as with unmodified RNA. In the example below, Trifectin was used to transfect HeLa cells with phosphorothioate LNA/DNA
chimeric antisense oligos targeting the microRNA miR-21. The psiCHECKTM-2 plasmid containing miR-21 recognition sites
cloned in the 3’-UTR of the Renilla Luciferase gene was used as a reporter system to measure miR-21 knockdown. Knockdown
of miR-21 results in an increase in the ratio of Renilla to firefly Luciferase (present as an internal control). The negative control
ASO (containing three base mismatches) does not alter Renilla Luciferase levels. Trifectin can be employed for all types of
antisense oligo applications, including traditional mRNA knockdown, splice-site blocking, and miRNA blocking.
Anti-miR21
T*+C*A*A*+C*A*T*+C*A*G*+T*C*T*+G*A*T*+A*A*G*+C*T*A
Figure 3a
Control Anti-miR21 3MUT
T*+C*A*A*+G*A*T*+C*A*G*+T*C*T*+C*A*T*+A*A*G*+G*T*A
+N = LNA bases, “*” = phosphorothioate bonds
Mismatched bases are indicated in blue
Figure 3
Introduction of miR21 ASOs into HeLa Cells
0 nM
10 nM
100
RLuc / FLuc Ratio
3a. Sequences of ASOs used. 3b. HeLa cells were plated in a 100mm
dish one day before transfection to give a 90% confluency 24 hours
later. Cells were transfected with 5 µg of psiCHECKTM-2 plasmid containing miR-21 binding sites in the 3’UTR of Renilla Luciferase. After
6 hours, cells were washed with 1XPBS, trypsinized, and replated at
2.3x104 cells/well in 48-well plates. Twenty four hours post-plasmid
transfection, the AS oligos were transfected at 50 nM, 25 nM or 10
nM with Trifectin or LipofectinTM in DMEM (serum-free). After six
hours, the cells were washed with 1XPBS and replenished with 250 µl
DMEM+10% FBS. Twenty-four hours after ASO transfection, cells were
lysed and analyzed for Luciferase activity with the Dual-LuciferaseTM
Reporter kit (DLRTM, Promega).
Transfection of Anti-miR21 ASOs in HeLa Cells
120
25 nM
80
50 nM
60
40
20
0
3MUT
Anti-miR21
3MUT
TriFECT in
Anti-miR21
Lipofectin
Figure 3b
Effective siRNA Dosage Determination
Once the transfection protocol has been optimized, it is useful to perform a dose response experiment to determine the minimum amount of siRNA or ASO needed to achieve the desired level of knockdown. Use of lower concentrations of oligonucleotides decreases the likelihood of triggering an off-target effect, such as stimulation of the innate immune system. Typical
dose response curves were shown above in Figures 1 & 2 using an anti-HPRT DsiRNA in T98G cells and HepG2 cells. Note that
these dose response curves are different even though the same DsiRNA and transfection reagents were employed and the
same maximal level of knockdown was seen.
When comparing the effectiveness of different transfection reagents and optimizing transfection protocols, it is advisable to
test the siRNA over a range of concentrations. Some reagents may give similar knockdown values when using a high dose of
the nucleic acid, but show marked differences in effectiveness at lower doses. Figure 4 illustrates this point in murine NIH3T3
cells. Trifectin and Reagent X were both equally effective when using a 10 nM concentration of the HPRT-1 DsiRNA, but at
lower doses Trifectin clearly performed better.
©2008 Integrated DNA Technologies
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I N T EG R AT E D DNA
T E C H N O LO G I E S
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REPORT
Comparing 2 Transfection Reagents
in 3T3 Cells
Dose-Dependant Reduction of HPRT-1 mRNA levels using Trifectin
Mouse 3T3 cells were reverse transfected with 10 nM, 1 nM and 0.1 nM
of DsiRNA HPRT-1, and a Negative Control Duplex with fixed amounts of
Trifectin or Reagent X in a volume of 600 µL. Cells were transfected at a
density of 7x104 cells per well in a 24-well plate in complete media. The
transfection mixture was left on the cells for 24 hours. RNA was isolated
and subjected to qRT-PCR to determine levels of HPRT mRNA. All HPRT
values were normalized to mouse ribosomal protein L23 RNA levels
(internal control) setting the negative transfection control at 100%.
% HPRT mRNA Levels
Figure 4
100%
80%
TriFECTin
Reagent X
60%
40%
20%
0%
10 nM
1 nM
0.1 nM
Neg Con
HPRT
Figure 4
Trifectin is Non-Toxic
Multiple cells lines (HCT116, 3T3, CHO, A549, HepG2, Hepa 1-6, HeLa) were treated with increasing amounts of Trifectin (2-6
µL) and examined for growth inhibition and cell death. There was no detectable cell death or decrease in cell growth even at
the highest concentration of Trifectin tested (data not shown). We have used as much as 10 µL of Trifectin in a 24-well plate
format and have not seen evidence of lipid-induced cell death even at this high dose.
Duration of Knockdown in Rapidly Dividing Cells
HeLa cells were transfected with Trifectin and anti-HPRT DsiRNAs (unmodified and nuclease-resistant 2’-O-Methyl modified)
targeting the HPRT-1 (hypoxanthine phosphoribosyltransferase 1) gene. Cells were harvested at 24-hour intervals and HPRT
mRNA levels were measured (Figure 5). Cells were healthy following transfection and showed 98-99% knockdown of HPRT at
days 1-3. Seven days post transfection the HPRT levels remained reduced by 80% and had still not returned to baseline by day
10.
Duration of Knockdown
Figure 5
Duration of Silencing Using DsiRNAs and Trifectin
HeLa cells were plated at 2.5x104 cells/well in a 24-well plate and transfected the next day using 1 µL of Trifectin and 10 nM of each DsiRNA.
Twenty-four hours later the media was changed. Cells were split on
days 2, 6 and 8. Cells were harvested at 24-hour intervals and RNA was
isolated; qRT-PCR was used to measure the levels of HPRT mRNA.
% HPRT mRNA Levels .
120
Unmod
2'OMe Mod
100
80
60
40
20
0
0
2
4
6
8
10
Days Post Transfection
Figure 5
Cell Lines Tested and Protocols
We have optimized transfection protocols for 14 commonly used cell lines in the R&D labs at IDT. For nine of these lines,
Trifectin was found to be the preferred transfection reagent. For five other lines, cationic lipids from other sources were found
to perform better. The cell lines tested and recommendations for primary and secondary transfection reagents are listed
below in Table 1.
Complete transfection protocols can be found in the Transfection Protocols Supplement.
©2008 Integrated DNA Technologies
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I N T EG R AT E D DNA
T E C H N O LO G I E S
TECHNICAL
REPORT
Table 1: Index of Cell Lines Tested at IDT and Transfection Reagent Recommendations
Cell Line
Cell Density For
24-Well Plate Reverse
Transfection
HeLa
Percent Knockdown
At 10 nM DsiRNA
5x104
99%
Primary
Recommendation
Secondary
Recommendation
Vol. Used
Vol. Used
TriFECTinTM
LipofectamineTM
RNAiMAX
1 µL
1 µL
TriFECTin
siLentFectTM
TM
T98G
6x104
96%
2 µL
0.6 µL
TriFECTin
siLentFectTM
TM
DU145
Hepa1-6
1.1x105
1.4x105
93%
94%
4 µL
0.5 µL
TriFECTinTM
LipofectamineTM
RNAiMAX
4 µL
2 µL
TriFECTin
siLentFectTM
TM
3T3
HCT116
A549
CHO
HepG2
7x104
90%
2x105
90%
6x10
89%
4
8x10
87%
4
1.1x105
88%
2 µL
0.5 µL
TriFECTinTM
LipofectamineTM
RNAiMAX
2 µL
1 µL
TriFECTinTM
LipofectamineTM
RNAiMAX
4 µL
2 µL
TriFECTinTM
LipofectamineTM
RNAiMAX
2 µL
1 µL
TriFECTinTM
TransIT TKOTM
4 µL
4 µL
Lipofectamine
RNAiMAX
TM
Neuro2a
RAT2
HEK-293
1.5x10
5
4x104
2x105
90%
siLentFectTM
2 µL
1 µL
94%
siLentFectTM
LipofectamineTM
RNAiMAX
94%
LipofectamineTM
RNAiMAX
siLentFectTM
1 µL
1 µL
TransIT TKO
TM
RAW
4x105
U87
1x10
5
80%
90%
None
2.5 µL
LipofectamineTM
RNAiMAX
siLentFectTM
2 µL
1 µL
siLentFectTM and siLentMersTM are registered trademarks of BioRad
LipofectamineTM RNAiMAX is a registered trademark of Invitrogen
TransIT TKOTM is a registered trademark of Mirus
Transfections were performed in 24-well format using 600 µl of medium. Protocols are outlined in the Transfection Protocols supplement.
©2008 Integrated DNA Technologies
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I N T EG R AT E D DNA
T E C H N O LO G I E S
TECHNICAL
REPORT
Technical Note
Contents and Storage
Handle under sterile conditions. Store at +4°C. Trifectin is guaranteed to be stable for a minimum of six months when
properly stored. Do not freeze or leave at room temperature for extended periods of time.
To qualitatively assess transfection efficiency, we recommend using the dye-labeled transfection control duplexes. To
quantitatively assess transfection efficiency, we recommend using the positive control duplexes. A variety of positive
controls are available.
Basic Protocol
Mix Trifectin with the siRNA or other nucleic acid, add to the cells, and incubate. Trifectin has low toxicity and it is not
necessary to remove the transfection cocktail before an extended incubation period. Use is compatible with both
forward and reverse transfection and in manual or robotic systems.
References
1. Conrad, A.H., Behlke, M.A., Jaffredo, T. and Conrad, G.W. (1998) Optimal lipofection reagent varies with the molecular
modifications of the DNA. Antisense Nucleic Acid Drug Dev, 8, 427-434.
2. Kim, D.H., Behlke, M.A., Rose, S.D., Chang, M.S., Choi, S. and Rossi, J.J. (2005) Synthetic dsRNA Dicer substrates enhance
RNAi potency and efficacy. Nat Biotechnol, 23, 222-226.
3. Rose, S.D., Kim, D.H., Amarzguioui, M., Heidel, J.D., Collingwood, M.A., Davis, M.E., Rossi, J.J. and Behlke, M.A. (2005)
Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res, 33, 4140-4156.
4. Amarzguioui, M., Lundberg, P., Cantin, E., Hagstrom, J.E., Behlke, M.A. and Rossi, J.J. (2006) Rational design and in vitro and
in vivo delivery of Dicer substrate siRNA. Nature Protocols, 1, 508-517.
©2008 Integrated DNA Technologies
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COMPANY
ABOUT IDT
Integrated DNA Technologies (IDT) is the largest supplier of custom nucleic acids in the U.S., manufacturing DNA and RNA
oligonucleotides (oligos) and serving the areas of academic research, biotechnology, and pharmaceutical development. IDT
products support applications including DNA sequencing, DNA amplification, expression profiling, microarray analysis, SNP
detection, gene quantification, and functional genomics. IDT is unique in its ability to accommodate even the largest orders
without compromising quality.
Founded by Dr. Joseph Walder in 1987, IDT’s development has been guided by an uncompromising approach to quality, a belief
in the value of good service, and a determination to minimize consumer costs. IDT’s production processes for both synthetic
oligos and consumables achieved ISO 9001:2000 certification in 2005 and ISO 13485:2003 certification in 2008.
IDT’s U.S. synthesis and research work is centered in Coralville, Iowa, in a 134,000 sq. feet (12,450 sq. meters) state-of-the-art
facility. West Coast customers are served from San Diego, where IDT began production in 2005. IDT established its European operating base in Belgium in 2006 and has constructed a new facility in Leuven, Belgium that incorporates its market-leading technology and manufacturing processes. Synthesis of an expanded product line began in this new facility in the spring of 2008.
IDT has grown by an average of 50% every year during the past 10 years, currently synthesizes and ships an average of 36,000
oligos per day, and has over 77,000 customers worldwide. The staff and scientists at IDT are dedicated to delivering the very best
in oligonucleotide synthesis, quality control, and customer service.
INNOVATION AND PRECISION IN NUCLEIC ACID SYNTHESIS
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I N T EG R AT E D DNA
T E C H N O LO G I E S
PROTOCOL
triFECTin
TM
The typical range of Trifectin needed is 1-6 uL per well in a 24-well culture plate (we have gone as high as 10 µL without any
observed cell death). We recommend starting with an siRNA concentration of 10 nM and titrating down (we usually study 10
nM, 1 nM, and 0.1 nM doses). With the small amount of RNA used in these studies, we do not alter the amount of lipid used
with changes in dose. Changes of the amount of lipid used should vary, however, with the scale of the transfection. For example, we usually use 0.5 µl of lipid/well when doing transfections in 96-well format. The transfections can be performed in a
forward or reverse format. Protocols for the optimization and use of Trifectin are provided below.
Optimizing The Amount Of Trifectin Needed In A Forward Transfection (24-Well Plate)
1. Determine the number of cells needed to plate to achieve 50-70% confluency, and plate the cells 12-16 hours prior to the
addition of the transfection reagent. A list of cell lines tested at IDT and the recommended amounts of reagent are shown
in Table 1 of the Trifectin Technical Report. The cell density listed in Table 1 is for the reverse transfection format, using a
24-well plate. When using the forward transfection format, reduce the cell numbers shown in Table 1 by half.
2. Dilute the siRNA (fluorescent control or HPRT control) duplex to 5 µM using an RNase-free annealing buffer (100 mM
potassium acetate, 30 mM HEPES pH 7.5).
3. Set up a series of tubes or a microtiter well plate containing 1-6 µL of Trifectin mixed with Opti-MEM® I to a final volume of
50 µL. This volume is enough for a single well of a 24-well plate.
4. Mix 48.8 µL of Opti-MEM® I and 1.2 µL of the 5 µM siRNA for each well of cells to be transfected plus one additional well
equivalent (e.g., For a 24-well plate, mix 1220 µL Opti-MEM® I [25 x 48.8] with 30 µL of siRNA [25 x 1.2]).
5. Incubate mixes for 5 minutes at room temperature.
6. Combine 50 µL of the siRNA-Opti-MEM® I mixture with 50 µL of the Trifectin-Opti-MEM® I, mix gently, briefly centrifuge and
incubate at room temperature for 10 minutes.
7. During the 10 minute incubation, remove media from cells and add 500 µL of antibiotic-free complete media. Trifectin is
compatible with the presence of serum during transfection.
8. Add mixture from step 6 dropwise onto cells. Rock the plate gently to mix. There is no need to remove the transfection
mixture from cells.
9. View cells under a fluorescent microscope at the appropriate wavelength or harvest cells 24 to 48 hours post transfection.
If using the HPRT positive control siRNA, isolate RNA and quantitate remaining levels of HPRT compared to a negative
control siRNA using qRT-PCR.
Example: Volumes and cell numbers needed to transfect HepG2 cells with
Trifectin in 24-, 48- and 96-well plates using the forward format.
96 well (1.38x104 cells in 150 µL total volume)
Reagent
TriFECTinTM /Opti-MEM® I
(Vol. in µL)
siRNA/Opti-MEM® I
(Vol. in µL)
Opti-MEM® I
11.5
12.2
Trifectin
1
-
siRNA 5 µM
-
0.3
48 well (2.75x104 cells in 300 µL total volume)
Reagent
TriFECTinTM /Opti-MEM® I
(Vol. in µL)
siRNA/Opti-MEM® I
(Vol. in µL)
Opti-MEM® I
23
24.4
Trifectin
2
-
siRNA 5 µM
-
0.6
24 well (5.5x104 cells in 600 µL total volume)
Reagent
TriFECTinTM /Opti-MEM® I
(Vol. in µL)
siRNA/Opti-MEM® I
(Vol. in µL)
Opti-MEM® I
46
48.8
Trifectin
4
-
siRNA 5 µM
-
1.2
©2008 Integrated DNA Technologies
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I N T EG R AT E D DNA
T E C H N O LO G I E S
PROTOCOL
triFECTin
TM
Optimizing The Amount Of Trifectin Needed In A Reverse Transfection (24-Well Plate)
1. Determine the number of cells needed to achieve 50-70% confluency. Twice that many will be needed per well. A list of
cell lines tested at IDT and the recommended amounts of reagent are shown in Table 1 of the Trifectin Technical Report.
The cell density listed in Table 1 is for the reverse transfection format, using a 24-well plate.
2. Dilute the siRNA (fluorescent control or HPRT control) duplex to 5 µM using an RNase-free annealing buffer (100 mM
potassium acetate, 30 mM HEPES pH 7.5).
3. Set up a series of tubes or a microtiter well plate containing 1-6 µL of Trifectin mixed with Opti-MEM® I to a final volume of
50 µL. This volume is enough for a single well of a 24-well plate.
4. Mix 48.8 µL of Opti-MEM® I and 1.2 µL of the 5 µM siRNA for each well of cells to be transfected plus one addition well
equivalent (e.g., For a 24 well plate, mix 1220 µL of Opti-MEM® I [25 x 48.8] with 30 µL of siRNA [25 x 1.2]).
5. Incubate mixes for 5 minutes at room temperature.
6. Combine 50 µL of the siRNA-Opti-MEM® I mixture with 50 µL of the Trifectin-Opti-MEM® I, mix gently, briefly centrifuge
and incubate at room temperature for 10 minutes.
7. During the 10-minute incubation, dilute trypsinized cells to achieve the appropriate cell number per well per 500 µL of
complete media without antibiotics.
8. Add the mixture from step 6 into the center of the well. Immediately add 500 µL of diluted cells to the well. Rock the plate
gently to mix. There is no need to remove the transfection mixture from cells.
9. View cells under a fluorescent microscope at the appropriate wavelength or harvest cells 24 to 48 hours post transfection.
If using the HPRT positive control siRNA, isolate RNA and quantitate remaining levels of HPRT compared to a negative
control siRNA using qRT-PCR.
Example: Volumes and cell numbers needed to transfect HepG2 cells with
Trifectin in 24-, 48- and 96-well plates using the reverse format.
96 well (2.75x104 cells in 150µL total volume)
Reagent
TriFECTinTM /Opti-MEM® I
(Vol. in µL)
siRNA/Opti-MEM® I
(Vol. in µL)
Opti-MEM® I
11.5
12.2
Trifectin
1
-
siRNA 5 µM
-
0.3
48 well (5.5x104 cells in 300 µL total volume)
Reagent
TriFECTinTM /Opti-MEM® I
(Vol. in µL)
siRNA/Opti-MEM® I
(Vol. in µL)
Opti-MEM® I
23
24.4
Trifectin
2
-
siRNA 5 µM
-
0.6
24 well (1.1x105 cells in 600 µL total volume)
Reagent
TriFECTinTM /Opti-MEM® I
(Vol. in µL)
siRNA/Opti-MEM® I
(Vol. in µL)
Opti-MEM® I
46
48.8
Trifectin
4
-
siRNA 5 µM
-
1.2
Opti-MEM® I is a registered trademark of Invitrogen
TriFECTinTM is a registered trademark of Integrated DNA Technologies
TriFECTinTM has not been tested for use with cell lines grown in suspension.
©2008 Integrated DNA Technologies
www.idtdna.com