 | | | | TransPass™ R1 Transfection Reagent | | | |
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Prices are in US dollars and valid only for US orders.
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Description:
A polyamine-lipid formulation designed specifically for the efficient transfection of siRNA in mammalian cell lines. It is a low toxicity reagent that in most applications does not require changing the medium after transfection.
NEB has determined that in addition to synthetic siRNAs, siRNA mixtures made with ShortCut® RNase III (NEB #M0245) can be very efficiently transfected in many mammalian cell lines with the TransPass™ R1.
Cell Lines Successfully Transfected:
A549, C6, CHO, COS-7, HEK293, NIH 3T3, HepG2, HCT116, HeLa, Jurkat, MCF-7, U20S, 3T3-L1, Huh-7, THP-1, preadipocytes, primary murine hepatocytes.
Table: TransPass siRNA Transfection Reagents Validated Cell Types
Figure 1: HCT116 cells transfected with Fluorescein-siRNA Transfection Control (NEB #N2100) using TransPass R1.
Figure 2: COS-7 cells were transfected with 6.0 nM Erk2 Kinase ShortCut siRNA Mix (NEB #N2003) (+) or 24 nM Lit28i Polylinker ShortCut siRNA Mix (NEB #N2014) (C) using TransPass R1 Transfection Reagent. Effective silencing of Erk2 Kinase is achieved at a low siRNA nanomolar concentration 48 hours post-transfection. Erk2 was detected in a Western blot of cell lysates using Cell Signaling Technology (CST #9107) Antibody. PKR loading control was detected using (CST #3072) Antibody.
Background:
RNA Interference (RNAi) is a method of post-transcriptional gene silencing in which the introduction into an organism of double-stranded RNA corresponding to a transcribed sequence results in degradation of the corresponding mRNA (for reviews, see references 2–9). Most mammalian cells treated with long dsRNA (over 30 bp) respond by a non-specific suppression of gene expression as well as apoptosis via the interferon response pathway (5).
In order to perform RNAi in mammalian cells, short dsRNAs (21–23 bp) are currently used because they are able to bypass this general non-specific response and achieve gene target-specific silencing via RNAi (6–8). To this end, synthetic oligo-ribonucleotides with 2 base 3´-OH overhangs (short interfering RNAs, siRNAs) are often used. One shortcoming of this approach is the difficulty of choosing effective 21 bp sites on the target RNA since the silencing effectiveness of siRNAs is highly dependent on the location of the corresponding target site (9).
Another approach, which mimics siRNA production in vivo by Dicer endonuclease (10), is to digest large dsRNA in vitro with ShortCut RNase III (see ShortCut RNAi Kit Manual, NEB #E2450) which results in complete conversion of large dsRNA into a size optimal for RNAi (18–25 bp) .
siRNA Transfection Guidelines:
The transfection of siRNA must be optimal in order to obtain maximum silencing of a target gene. Optimizing the following parameters may be necessary in order to maximize the transfection efficiency for a particular cell line: cell density at the time of transfection, amount of transfection reagent, amount of siRNA and culture incubation time before analysis.
For consistent results, it is important to maintain healthy proliferating cells that are regularly passaged.
It is important that NO heparin and NO antibiotics/antimycotics in the growth medium during transfection.
Reaction & Storage Conditions
Concentration:
2.5 mg/ml
Storage Temperature:
4°C
Do not freeze.
Notes
General notes:- It is important to maintain healthy cells. Some cell lines become more sensitive to transfection agents after a large number of passages. It is advisable to use cells subjected to a similar number of passages to ensure reproducible transfection results in different experiments.
- It is recommended that control transfections be performed by varying cell confluence and using different amounts of TransPass Transfection Reagent. In general, low cell density or too much transfection reagent increase the risk of cell toxicity. The medium may be replaced 24 hours after transfection with fresh complete medium to increase cell viability.
In order to easily estimate the efficiency of transfection of particular cell lines use Fluorescein-siRNA Transfection Control (NEB #N2100S). - TransPass R1 Transfection Reagent has been used to successfully transfect siRNAs in many cell lines including: A549, C6, CHO, COS-7, HEK293, NIH 3T3, HepG2, HCT116, HeLa, Jurkat, MCF-7, U2OS, 3T3-L1 preadipocytes.
- TransPass R1 Transfection Reagent has been developed for the transfection of siRNA, siRNA mixtures, in vitro transcribed RNA hairpins etc. It has not been optimized for plasmid DNA transfection. The transfection efficiency of plasmid DNA varies from cell line to cell line. If you wish to transfect both DNA and siRNA in the same transfection and the efficiency of DNA transfection with TransPass R1 Transfection Reagent is not sufficiently high for your application, you can use the protocol 2. For efficient DNA transfection we recommend the use one of the DNA transfection reagents TransPass D1 (NEB #M2553S, and TransPass D2 NEB #M2554S).
- TransPass R1 Transfection Reagent can be safely stored for 6 months at 4°C. Tightly cap the tube to prevent evaporation.
Protocols
Protocols for TransPass™ R1 Transfection Reagent
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Controls:
Each lot of TransPass R1 Transfection reagent is tested by NEB for efficient delivery of labeled synthetic siRNA into different cell lines and for endogenous gene silencing using ShortCut siRNA Mixes (Figures 1,2).
References
- Fire, A. et al. (1998) Nature, 391, 806-811.
- Bass, B.L. (2000) Cell, 101, 235-238.
- McManus, M.T. and Sharp, P.A. (2002) Nature Reviews, Genetics, 3, 737-747.
- Stark et al. (1998) Ann. Rev. Biochem, 67, 227-264.
- Elbashir, S.M. et al. (2001) Nature, 411, 494-498.
- Sharp, P.A. and Zamore, P.D. (2000) Science, 287, 2431-2433.
- Brummelkamp, T.R. et al. (2002) Science, 296, 550-553.
- Holen, T. et al. (2002) Nucleic Acids Res., 30, 1757-1766.
- Bernstein, E. et al. (2001) Nature, 409, 363-366.
Companion Products
Erk2 Kinase ShortCut® siRNA Mix
Fluorescein-siRNA Transfection Control
Lit28i Polylinker ShortCut® siRNA Mix
Luciferase Cell Lysis Buffer
ShortCut® RNAi Kit
siRNA Marker
TransPass™ R2 Transfection Reagent
Legal
Research Use Assurance:
Manufactured by Mirus Bio Corp. and covered by its patents and patents pending.
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