Procedure for the Generation of dsRNA for use in RNAi
1. Design polymerase chain reaction (PCR) primers that will amplify 600-800 bp of sequence corresponding to the message of interest. Both primers should contain the following T7 RNA polymerase binding site at the 5’ end:
GAA TTA ATA CGA CTC ACT ATA GGG AGA.
Here's one for T3:
AAT TAA CCC TCA CTA AAG GGA GA
I usually follow the T7 RNA polymerase binding site with 18 nucleotides specific for the mRNA of interest to create an oligo that is 45 bp in length.
2. Use these primers in a 100 ul PCR to GENErate a 600-800 bp product. 5 ul of the reaction should be analyzed by agarose gel electrophoresis. If a robust band of the appropriate size is not apparent on the gel, reamplify the reaction using a second PCR.
3. Purify the PCR product using a High Pure PCR Product Purification Kit (Roche or Qiagen).
4. Add the appropriate amount of purified product to a transcription reaction to produce RNA. I use the MaxiScript (or MegaScript) T7 transcription reaction from Ambion which produces >50 ug of RNA. For injection into Drosophila embryos, you will need 85 pL of 5 uM (roughly 1.7 ug/ul) dsRNA. There is no need to anneal the RNA produced in this reaction as the two RNA strands self anneal during synthesis.
It is a straightforward transcription reaction from a PCR product. The dsRNA generated this way is relatively stable and can be analyzed by agarose gel electrophoresis.
Note: dsRNA needs to be spun down or filtered through a Co-Star to prevent needle clogging.
Protocol from Zipursky Lab, UCLA (Jim)