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RNAworld 11-02-2010 12:11 AM

Reverse Transcription (RT)
 
I am trying to get reverse transcription and dideoxy sequencing on my RNA. but since my RNA has strong secondary structure, the reverse transcriptase cannot get through it. I am using AMV at 52oC.

Also, I see a lot of background other than the full length product and unextended primers in my control lane, . anyone can help?

danfive 11-02-2010 04:20 PM

Re: Reverse Transcription (RT)
 
Quote:

but since my RNA has strong secondary structure, the reverse transcriptase cannot get through it
That is a big assumption, and the least likely cause. The secondary structure can be there but really never seen it stop an RT-PCR.

Try:
reducing amount of RNA (likely cause of background)
Make sure RNA is best quality you can get (RNAse free, desalted, fresh)
1 primer only for transcription (low concentration so it gets used up)
reduce primer-dimer problem in PCR (reduce concentration of both primers, optimize #cycles, PCR-annealing temp, extension time etc).
____________________________________________--
After doing some or all of the above and if still in need of optimization:
you can pre-melt RNA (melting the 2nd structure really 95C for 30-50seconds) with primer (mixed/vortexed in RT rxn buffer only); very quick chill on ice, followed by adding rest of RT rxn mix and then beginning your RT rxn.
--A specific primer annealing should be facilitated and should improve your final results.

Good luck.

RNAworld 11-02-2010 10:20 PM

Re: Reverse Transcription (RT)
 
I will try that tomorrow! Thanks! will there be a big difference if i pre-melt RNA in water and in RT rxn buffer?

danfive 11-03-2010 01:31 AM

Re: Reverse Transcription (RT)
 
Quote:

Originally Posted by RNAworld (Post 423551)
I will try that tomorrow! Thanks! will there be a big difference if i pre-melt RNA in water and in RT rxn buffer?

Use RT or PCR buffer, you need the buffer for quality control/protection from hydrolysis.


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