USER GUIDE

Silencer siRNA Construction KitCatalog Number AM1620Publication Number 1620M Revision Rev. E

For Research Use Only. Not for use in diagnostic procedures.Information in this guide is subject to change without notice.

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NOTICE TO PURCHASER: LIMITED USE LABEL LICENSE: Research Use OnlyThe purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

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Contents

3Silencer siRNA Construction Kit User Guide

Silencer siRNA Construction Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Reagents provided with the kit and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Materials not supplied with the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Silencer siRNA Construction Kit instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8siRNA design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Transcription template preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10dsRNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12siRNA preparation/purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13siRNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Transfecting mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Using the Control DNA supplied with the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Low yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Transfected siRNA does not reduce gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Gel analysis shows multiple bands or incorrect siRNA size . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

APPENDIX A Supplemental Information . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Additional procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Gel analysis of siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Gel analysis of DNA oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Gel purification of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Assessing mammalian cells following siRNA transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Additional recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2710X TBE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272% agarose gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2712% acrylamide gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Nondenaturing gel loading buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28RNase-free water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Related products available from Life Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

APPENDIX B Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

4 Silencer siRNA Construction Kit User Guide

Contents

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

5Silencer siRNA Construction Kit User Guide

Silencer siRNA Construction Kit

IMPORTANT! Before using this product, read and understand the information in the Safety appendix in this document.

Introduction

Background Post-Transcriptional Gene Silencing and RNA interference (RNAi) are terms describing the specific suppression of genes by complementary dsRNA (for a recent review, see Sharp 2001). Although the mechanism by which dsRNA suppresses gene expression is not entirely understood, experimental data provide important insights. In non-mammalian systems such as Drosophila, it appears that longer dsRNA is processed into 2123 nt dsRNA (called small interfering RNA or siRNA) by an enzyme containing RNase III motifs (Bernstein et al., 2001, Grishok et al., 2001, Hamilton and Baulcombe 1999, Knight and Bass 2001, Zamore et al., 2000). The siRNA apparently then acts as a guide sequence within a multicomponent nuclease complex to target complementary mRNA for degradation (Hammond et al., 2001).

Long dsRNA is routinely used in non-mammalian cells and organisms to effect gene silencing. Mammalian cells, however, have a potent antiviral response pathway that induces global changes in gene expression when dsRNA molecules longer than 30 nt are introduced into cells (Stark et al., 1998, Manche et al., 1992, Minks et al., 1979). The antiviral response makes it difficult to distinguish target-specific effects of long dsRNA from the general antiviral response. siRNAs, comprised of 21-mer dsRNAs, do not trigger the antiviral response, making it possible to perform gene silencing experiments in mammalian cells (Elbashir et al., 2001, Caplen et al., 2001, Hutvagner et al., 2001, Jarvis and Ford 2001).

siRNA synthesis by in vitro transcription

Whereas most RNAi experiments in Drosophila, C. elegans, and other non-mammalian cells have used in vitro transcribed RNA molecules, early siRNA experiments in mammalian cells used chemically synthesized RNA oligomers. Chemically synthesized siRNAs were probably used because there are no sequence limitations for chemical synthesis. In contrast, in vitro transcription using T7 RNA polymerase requires that the first 2 nucleotides of the RNA transcript be GG or GA to ensure efficient synthesis (Milligan 1987). Requiring a GG or GA at the 5' ends of both the sense and antisense strands of an siRNA in addition to the required 3' terminal UU greatly reduces the number of potential target sites for siRNA experiments. This constraint essentially elimin