MessageMuter™ shRNAi Production Kit

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Last modified: 2008-07-15 14:57:15

RNA interference (RNAi) is a powerful technique for elucidation of gene function. Short hairpin RNA (shRNA) (Figure 1), produced by chemical synthesis, in vitro transcription, or expressed in vivo, has been shown to silence genes as effectively as short dsRNAs.

The MessageMuter™ shRNA Production Process

The MessageMuter shRNA Production Kit utilizes a simple and unique 3-step process (Figure 2) that yields transfection-ready shRNA in about 4 hours. A 17-base T7 Promoter Oligo is annealed to a DNA oligo designed by the user using detailed instructions provided. The ends of the annealed duplex are "filled-in" using the Exo-Minus Klenow to obtain a linear dsDNA with a T7 RNAP promoter at its 5'-end. Following in vitro transcription, the RNA produced spontaneously forms a hairpin structure (shRNA). Following clean-up, the shRNA is ready for transfection into cultured cells. The MessageMuter shRNA kit provides reagents to produce 10 shRNAs in sufficient quantities for hundreds of RNAi experiments.

1A		Figure 1 Comparison of short hairpin RNA (shRNA) and siRNA directed against the same firefly luciferase sequence. 1A. The shRNA produced using the MessageMuter™ shRNA Production Kit contains a sequence homologous to the target mRNA (sense sequence), a "loop" region and a sequence complementary to the target sequence (anti-sense sequence). 1B. An siRNA targeting the same 21-base sequence of firefly luciferase mRNA.
1B

Figure 2 Overview of the method used to produce shRNA using the MessageMuter™ shRNA Production Kit. All reagents are supplied except a 60- to 76-base DNA oligo designed by the user as specified in the kit.

Last modified: 2008-05-14 16:46:46

Benefits

• Significantly lower cost per RNAi experiment than chemical synthesis of dsRNA.
• Faster and easier than constructing short dsRNA by other strategies
• No need for annealing of sense and anti-sense RNA strands as required by other in vitro transcription-based methods
• High potency shRNA is produced in about 3 hours
• Each reaction produces enough shRNA for 100's of RNAi experiments

Figure 3. Gene silencing by shRNA made using the MessageMuter™ shRNA Production Kit. The effect of shRNA targeted against firefly luciferase expression ("shRNA-luc") in a variety of cultured mammalian cells is presented as a ratio of firefly luciferase activity to renilla luciferase activity. 10 nM shRNA-luc suppressed firefly luciferase expression by ~90% relative to renilla luciferase in all cell types tested.

Use of dsRNA for RNA interference by for-profit organizations requires a license from the Carnegie Institution of Washington. For-profit institutions should contact Gloria Brienza of the Carnegie Institution of Washington, 1530 P Street, N.W., Washington, D.C. 20005-1910. Email: gbrienza@pst.ciw.edu.

Last modified: 2008-05-14 16:46:46

Protocols for: MessageMuter™ shRNA Production Kit

(catalogue number MM031110)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46

References

1. McManus, M.T. (2002) RNA 8 , 842.
2. Paddison, P.J. et. al. (2002) Genes & Development 16, 948.
3. Yu, J-Y et. al. (2002) Proc. Natl. Acad. Sci. USA 99 (9), 6047.
4. Meis, J.E. (2003) EPICENTRE Forum 10 (2), 1.
5. Meis, J.E. (2004) EPICENTRE Forum 11 (1), 1.

Last modified: 2008-05-14 16:46:46