Designing and Preparing dsRNA

Ordering dsRNA Primers

• If available, choose primers from Harvard DSRC site http://www.flyrnai.org/cgi-bin/RNAi_gene_lookup_public.pl
• Add T7 RNA Polymerase sites to the 5' of each primer (TAATACGACTCACTATAGG)
• Record primer and amplicon (DSRC)

Preparing dsRNA

Template Preparation

• set up a 250 uL PCR reaction
  • 25 uL 10X KOD buffer
  • 25 uL dNTPs
  • 50 uL Primers (sense and antisense combined and diluted to 1 uM. 2 uL of each primer plus 196 uL of water)
  • 15 uL MgCl (this can be adjusted to optimize PCR conditions)
  • Template (previously prepared PCR product or 1 uL of cDNA)
  • 2 uL KOD polymerase
  • 132uL Water (bring up to 250 uL)
  • Run using TD-KOD program
• transfer PCR product to a clean tube. Add 25 uL of 3M NaAcetate (pH 5.2) and 500 uL ice cold ethanol (stored at -20).
• Incubate on ice for >20 min.
• Centrifuge 5 min at 4C on high in eppendorf centrifuge.
• Carefully aspirate supernatant. Add 1 mL 70% ethanol and centrifuge again.
• Aspirate supernatant and let pellet air dry.
• Resuspend in 20 uL EB and measure concentration by nanodrop.

RNA Synthesis

• Prepare RNA synthesis reaction (RiboMAX Large Scale RNA Preparation Kit):
  • 20 uL T7 Buffer
  • 30 uL rNTPs (prepare by combining equal volmes of rNTP together)
  • 10 uL Enzyme mix
  • 10 ug PCR product
  • water to bring volume up to 100 uL
• Place in PCR machine and incubate overnight at 37C (37 hold program)

Quantify RNA

• Dilute RNA 10x in loading dye
• Load 1 and 3 uL of diluted RNA, plus 4 or 8 uL of 1 kB Plus DNA Ladder
• Using ImageJ, quantify ladder and dsRNA bands and calculate concentration. See Using ImageJ to Quantify Bands. Check that the dilutions are close to 3x apart in signal intensity.
• Label RNA with concentration and store at -20

• see PMID 18388942, PMID 18265350 , PMID 11752672 and http://www.flyrnai.org/DRSC-PRS.html