Design and Testing of siRNAs
Step ONE
Develop a transfection protocol for the cells being evaluated.
Transfect target cells with several concentrations of an siRNA specific to GAPDH (included as a control with the Silencer™ siRNA Construction Kit).
48 hours after transfection, measure the reduction in GAPDH protein or mRNA levels compared to untransfected cells.
Vary the concentration of the transfection reagent to optimize the transfection conditions.
Step TWO
Select siRNA target sites for the gene being evaluated.
Begin with the AUG start codon and scan the length of the gene for AA sequences. Record the AA and the 3' adjacent 19 nucleotides as potential siRNA target sites.
Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate any target sequences with significant homology to other genes.
Select 4 of the qualifying target sequences for analysis. Try to select target sequences along the length of the gene to test whether the 5', 3', or medial portions of mRNAs are more susceptible to siRNA induced degradation.
Step THREE
Prepare siRNAs using the Silencer™ siRNA Construction Kit (below is a brief outline of the procedure).
Order two 29mer DNA oligonucleotides for each siRNA being tested. Hybridize the DNA oligonucleotides to the T7 Promoter Oligonucleotide and prepare the dsDNA template using DNA polymerase.
Add each template to a separate transcription reaction and incubate for 2-4 hr at 37°C. Mix the transcription reactions corresponding to the opposite strands of each siRNA and incubate overnight at 37°C.
Treat the transcription reactions with DNase and a single-strand specific RNase to eliminate DNA templates, siRNA leader sequences, and unhybridized RNA.
Column purify the siRNA.
Step FOUR
Transfect mammalian cells with gel-purified siRNA using the protocol developed in Step 1 above.
Step FIVE
Evaluate expression level of the target mRNA or protein 24, 48, and 72 hr post-transfection.
Step SIX
Design and test 1 or 2 negative control siRNAs.
Negative control siRNAs should have the same nucleotide composition as the most active siRNA. It should lack significant sequence homology to the genome of the cells being used (i.e. scramble the nucleotide sequence of the most active siRNA and then do a homology search to make sure it lacks homology to genes in the organism).