3 replies to this topic

[talianka]

Dear colleagues,

I would like to ask, whether it is necessary to precipitate the RNA (or even using phenol:chloroform extraction) after treatment with DNase , or I can directly use the DNase treated mixture for cDNA synthesis (as some manufacturers, e.g. Fermentas) recommend? I guess, by precipitation, I won't get rid of inactivated enzyme anyway, and by precipitating, I will only eliminate salts and divalent cations. My experience with using cDNA reverse transcribed from precipitated RNA was, that after PCR on the cDNA and gel electrophoresis, I got a strong band (or rather signal) sitting in the gel  socket (non-migrating) - please see the picture. I also amplified the fragment of my interest, but I have no idea what is this non-migrating "thing" - I just worry that it could be the DNA bound to something that doesn't migrate, and possibly preventing the amplification. I would like to have the good RNA for subsequent qRT-PCR applications and find a compromise between the RNA quality and number of steps necessary, because each step introduces some kind of bias
One more question - if you want to test the efficiency of various cDNA reverse transcription kits, is the only way to test it by qRT-PCR?

Many thanks in advance!

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[SarahG]

It is not necessary to remove the DNase as long as you inactive it prior to cDNA synthesis. This is typically done by heating at 65-70 C for 10-30 minutes. You also need to add EDTA to prevent the RNA from hydrolyzing during heating.

That strong signal you are getting on your gel that just stays in the well, is this from a sample that was not DNase treated? My guess is that it is genomic DNA. If this sample was DNase treated, you might need to use more DNase.

[talianka]

Many thanks for the reply.
Samples on the gel are PCR products amplified from cDNA. This cDNA comes from the reverse transcribed RNA that was DNaseI treated before reverse transcription. If it was DNA, I am just wondering why it would not migrate in the gel as well?

[Trof]

Other option is to use kit like DNAfree from Ambion (or whatever Life Technologies it now is) that is inactivated by some sandy reagent included so you just need to centrifuge it and pipette the supernatant out.