siRNA protocols

Our current strategy with siRNA is to synthesis relatively small amounts enzymatically and use these to test for efficiency by western blotting. This allows us to test several candidate siRNAs quite cheaply. The yields are good enough for reasonable amounts of experimental work, but for larger use we get successful siRNAs synthesised chemically by Dharmacon.

Design The Tuschl lab siRNA users guide gives information about how to choose the basic target sequence. For enzymatic synthesis, the target sequences for the preparation of T7 polymerase transcribed siRNA are of the form G(N17)C. The first and last nucleotide constraints are required because T7 polymerase puts a G at the start of all transcripts. RNAs are synthesised from templates that are double stranded in the region of the T7 promoter and single stranded in the region encoding the RNA of interest. Such templates are generated by annealing a 20-mer T7 promoter primer oligonucleotide to a 40-mer target oligonucleotide, one for the sense strand and one for the antisense strand. Transcription from these templates will generate complementary RNAs. Once annealed, the siRNA will be double stranded for 19 nt with 2 NT 3' overhangs, always UU in the sense strand RNA.

siRNA synthesisi. Annealing template For this you need to prepare two reactions: one for the sense oligo and one for the antisense oligo. Dissolve oligo at 100 μm in water and combine oligo with T7 promoter oligo at 5 uM each in 90 mM Tris pH8.0. 90μl 90mM Tris, pH 8.0

5μl oligo

5μl T7 promoter oligo (5'-GGTAATACGACTCACTATAG-3')Heat at 95℃ for 3min.

Snap cool on ice.

ii. Small scale transcription reaction Set up two reactions. One with the sense template and one with the antisense template.40μl water

5μl 10x T7 Buffer*

2μl 25mM NTPs (1mM final)

1μl annealed template mix (100nM final)

2μl T7 RNA polymerase (50U/μl)

Incubate at 37℃ for 2h.

Add 1ul (1U/ul) DNase I (RNase Free)

Incubate at 37℃ for 15min *10x T7 buffer: 0.4M Tris pH 8, 60mM MgCl2, 50mM DTT, 10mM spermidine, 0.1% Triton X-100, 500ug/ml BSA. Prepare without BSA, filter through 0.2μm filter, add BSA, correct volume.