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.
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.
i. 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 uM in water and combine oligo with T7 promoter oligo at 5 uM each in 90 mM Tris pH8.0.
90ul 90mM Tris, pH 8.0
5ul T7 promoter oligo (5'-GGTAATACGACTCACTATAG-3')
Heat at 95oC 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.
5ul 10x T7 Buffer*
2ul 25mM NTPs (1mM final)
1ul annealed template mix (100nM final)
2ul T7 RNA polymerase (50U/ul)
Incubate at 37oC for 2h.
Add 1ul (1U/ul) DNase I (RNase Free)
Incubate at 37oC 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 0.2um filter, add BSA, correct volume.
iii. Generate siRNA duplexes
Combine sense and antisense transcription reactions.
Heat at 95oC for 5min.
Incubate at 37oC for 1h.
Precipitate by adding 6.7ul 3M sodium acetate pH 5.2 + 250ul ethanol.
(Note that there is no need to chill precipitations as there is plenty of RNA and chilling can cause precipitation of undesirables)
Centrifuge in benchtop microfuge at room temperature for 5min.
Wash once with 70% ethanol
Dissolve in 50ul water.
We have used Oligofectamine transfection successfully with a range of mammalian cultured cells. The protocol is that derived and optimised by the Tuschl lab and can be found in the very useful siRNA users guide.
ii. Calcium phosphate
Day 1 Plate HeLa cells at 75000 cells/ml in DMEM with 10% FCS.
Day 2 Transfect in late afternoon/evening. Mix siRNA with filter-sterilised 0.25 M CaCl2 then with filter-sterilised BBS (50 mm BES, pH 6.95, 280 mm NaCl, 1.5 mm Na2HPO4). Leave 15-20 min then add dropwise to plates whilst swirling. Remove plate to a 3% CO2 incubator.
For a 10cm plate with 10 ml medium, use 500ul CaCl2, 500ul BBS and 10ul siRNA.
Day 3 Wash plate once with PBS then add fresh medium and return to 5% CO2 incubator for 1 hour. Wash once more with PBS and replace with fresh medium again.
Day 4 Harvest.
Oligofectamine works very well but is also very expensive. We have had seemingly identical results using calcium phosphate, at least in HeLa cells. This has the obvious advantage of being much cheaper.
In our hands, the yield of siRNA from the small scale synthesis is 0.5-2.5ug. This provides sufficient reagent to transfect 5-10cm plates or 25 wells of 6-well dishes. The reaction should scale up with no loss of yield.
The RNAs can be visualised on a 4% agarose gel. We recommend using low melting temperature agarose and loading samples without sample buffer into dry wells. Run the gel for 5-10 min with sufficient running buffer to generate a current through the gel without covering the top. Once the samples have entered the gel, cover the gel with buffer and continue the run. Sample buffer in a separate lane should be used to assess progress of the run.
All reagents are purchased as molecular biology grade and RNase free. Fingertips and equipment used for DNA preparations involving RNase are the most likely sources of contaminating RNase. The best way of ensuring an RNase free environment is to use plasticware, including tips, that have never been touched by human hands. Autoclaving is probably not a very effective method of removing RNase. Change gloves regularly to protect against cross contamination.
The above procedure is based upon published methods from:
1. Milligan,J.F. and Uhlenbeck,O.C. (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol., 180, 51-62.
2. Elbashir,S.M., Harborth,J., Lendeckel,W., Yalcin,A., Weber,K. an Tuschl,T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494-498.
3. Donze,O. and Picard,D. (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res., 30, e46.
4. Chen,C.A. and Okayama,H. (1988) Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques, 6, 632-638.