dsRNA synthesis

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Thu Jul 16 15:29:01 EST 1998


Earlier I asked:

>Has anyone synthesized both RNA strands in a single reaction?
>...

>Has anyone tried simultaneous double-strand RNA synthesis in vitro (or have
>any other comments on this)?

Thank you to those who replied.  Here is a summary of the responses I
received (as of Thursday 16 July 1998):


From: Michael Hengartner <hengartn at cshl.org>

>I think that Craig Mello tried, and that he actually found this to be the
>most effective way of preparing the dsRNA!


From: Neville Ashcroft <ashcrofn at ncifcrf.gov>

>I've tried synthesising both strands for dsRNAi in the same reaction.  I
>normally prepare the template by PCR - this time I used oligos that had T7
>sequences added to both the forward and reverse primers.  I ran the
>synthesised RNA with controls on a gel.  The dsRNA gave a tighter band
>compared to just ssRNA, so it looked like the synthesis worked.  Indeed,
>the dsRNAi gave a stronger phenotype than just the antisense RNA alone.


From: Edward Kipreos <ekipreos at cb.uga.edu>

>I have synthesized both T7 and T3 reactions at the same time using Ambion's
>MegaScript kits (the buffers are the same). With the Ambion kits, the RNA
>is so viscous (3-6 mg/ml) that we have to dilute the RNA a minimum of 1:4
>and often 1:10 to load needles, especially after annealing the two strands.
>To decrease the viscosity of annealed products, we diverge from Andy Fire's
>protocol by heating at 95 deg. for 5 min. and then annealing at 70 deg. for
>10 to 20 min. This allows more one-to-one priming without multimer
>formation and allows us to use a more concentrated dilution for injection.
>We found that synthesis of both reactions simultaneously gives us the same
>decrease in viscosity (without any annealing step). All conditions give
>RNAi effects, the more concentrated dilutions seem to help with troublesome
>genes.


From: Jeb Gaudet <jeb.gaudet at utoronto.ca>

>Just wondering why you want to do the reactions simultaneously.  Just as a
>time saver or is there another consideration?

Mostly as a time saver, as well as possibly a reagent saver; if one 20 ul
reaction (with both T3 and T7 RNA polymerase) will suffice, why do two
reactions (with each polymerase separately)?


By the way, I went ahead and did a set of three reactions (T3 RNA pol, T7
RNA pol, and both RNA pols) and found that:
(1) dsRNA was present in the T3+T7 transcription reaction before
denaturation/renaturation,
(2) the apparent levels of dsRNA increased slightly after
denaturation/renaturation, and
(3) the levels of dsRNA after denaturation/renaturation were comparable
whether the strands were synthesized in two separate reactions or in one
(actually combining the separate reactions gave slightly more dsRNA in my
one experiment).

Best wishes,
Bill Morgan

P.S. Here's my original posting:
>Dear Worm-Breeders,
>
>Has anyone synthesized both RNA strands in a single reaction?
>
>I'm preparing to synthesize double-stranded (ds) RNA for RNA-mediated
>inhibition experiments.  The "RNAi info from the Fire Lab" manual describes
>the synthesis of each RNA strand separately, with a subsequent annealing
>reaction to produce dsRNA.  I'd like to synthesize both RNA strands
>simultaneously in a single reaction using both T3 and T7 RNA polymerases.
>Both polymerases work under identical conditions (according to Promega) and
>I've cleaved the DNA templates at sites flanking the T7 and T3 promoters
>(either, BssHII or PvuII, depending on the pBluescipt vector), so it seems
>like it should work.
>
>Has anyone tried simultaneous double-strand RNA synthesis in vitro (or have
>any other comments on this)?
>
>If there's sufficient response and interest, I'll post a summary to the group.
>
>Bill Morgan


William R. Morgan
931 College St.
Department of Biology			Phone:  330-263-2026
The College of Wooster                  FAX:    330-263-2378
Wooster, OH  44691                      E-mail: wmorgan at acs.wooster.edu





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