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  • TransPass™ R2 Transfection Reagent

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    * This reagent has two solutions: Solution A - 0.5 ml, Solution B - 1.0 ml

    The TransPass™ R2 Transfection Reagent is a two component non-liposomal reagent combination developed specifically for efficient transfection of siRNA in a variety of mammalian cell lines that include “difficult to transfect" cells such as primary cells, endothelial cells, lymphocytes, muscle cells, etc. It shows reduced levels of cell toxicity as compared to cationic liposome-based reagents.

    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™ R2.

    Efficient delivery of labeled siRNA and silencing of endogenous genes has been achieved in many cell lines including:

    A549, COS-7, HEK293, HepG2, NIH3T3, HeLa, OVCAR-3, HUVEC, HMVEC, THP-1, B-lymphoma cells, vascular smooth muscle cells, human lens epithelial cells.

    Table:
    TransPass siRNA Transfection Reagents Validated Cell Types

    Figure 1:
    OVCAR cells transfected with a Cy3-labeled siRNA using TransPass R2.
    Figure 2:
    HeLa cells were transfected with different concentrations of p38 MAPK ShortCut siRNA Mix (+) or 24 nM eGFP ShortCut siRNA Mix (C) using TransPass R2 Transfection Reagent. Effective silencing of p38 MAPK is achieved at low siRNA nanomolar concentration 48 hours post-transfection. p38 MAPK was detected in a Western blot of cell lysates using Cell Signaling Technology (CST #9217) Antibody and PKR loading control was detected using (CST #3072) Antibody.

    Advantages and Features

    Features


    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 1,3-9). 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 (7).

    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)(2).

    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 are in the growth medium during transfection.

    Properties and Usage

    Storage Temperature

    4°C

    Notes

    1. 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.
    2. It is recommended that control transfections be performed by varying cell confluence (40-90%). In general, low cell density or too much transfection reagent increase the risk of cell toxicity. The transfer medium may be replaced 2 hours after transfection with fresh complete medium to increase cell viability.
    3. In order to easily estimate the efficiency of transfection of particular cell lines use Fluorescein-siRNA Transfection Control (NEB #N2100).
    4. TransPass R2 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 plasmic DNA varies from cell line to cell line. For efficient DNA transfection we recommend the use of one of the DNA transfection reagents: TransPass D1 (NEB #M2553) or TransPass D2 (NEB #M2554).
    5. TransPass R2 Transfection Reagent can be safely stored for 6 months at 4°C.

    References

    1. Fire, A. et al. (1998). Nature. 391, 806-811.
    2. Bass, B.L. (2000). Cell. 101, 235-238.
    3. McManus, M.T. and Sharp, P.A. (2002). Nature Reviews. Genetics. 3, 737-747.
    4. Stark et al. (1998). Ann. Rev.. Biochem. 67, 227-264.
    5. Elbashir, S.M. et al. (2001). Nature. 411, 494-498.
    6. Sharp, P.A. and Zamore, P.D. (2000). Science. 287, 2431-2433.
    7. Brummelkamp, T.R. et al. (2002). Science. 296, 550-553.
    8. Holen, T. et al. (2002). Nucleic Acids Res. 30, 1757-1766.
    9. Bernstein, E. et al. (2001). Nature. 409, 363-366.
    10. Xiao, J. et al. (2009). RNA. 15, 984-991.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.
    1. Cellular Imaging and Analysis FAQs
    1. TransPass R2: Plasmid DNA and siRNA Transfection Protocol
    2. TransPass R2: siRNA Transfection Protocol

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