Reverse transcription problems - (Jul/08/2005 )

Hello,
I got many problems with reverse transcription, and some questions:
1) How common is to treat RNA with DNAse I/RNase free to avoid genomic DNA contamination? How do you inactivate it ?
2) Have you determined the optimal Mg concentration of the MMLV reverse transcriptase?
3)What happens if you add more RNA template than recomended trying to improve cDNA yield? What is the concentration that you got?

Hello!

I may not be much help cos I don't use MMLV. I don't add DNase to my samples after extraction. I use Trizol, and stay well away from the DNA phase when I'm taking the supernatant (i.e. leave 40% behind, and take 60% of supernatant that is supposed to contain RNA). Seems to work well for me.

G

I will have in mind to stay as far as I can from the lower phase when taking the aquous phase. One more question about trizol extraction: do you use the isopropanol at room temperature? I use it freeze. I am suspecting that cold isopropanol is helping co-precipitate the remaining genomic DNA in the aquous phase.

The trizol protocol calls for room temp isopropanol... use room temp isopropanol.

Ok, Now I am clear enough about isopropanol temperature. Do you know why I did use it freeze? because it has always been performed like that here in the lab. What a stupid I was!!

[/I]Hey there! I'm going to try to answer your questions in italics under each question, since you've got lots of replies but no answers...

1) How common is to treat RNA with DNAse I/RNase free to avoid genomic DNA contamination? How do you inactivate it ?
I think its common and INCREDIBLY important. When I don't I always get residual DNA contamination in my noRT lane for the PCR, and sorry, but that's just unacceptable. I'm extremely careful during Trizol isolation too, so I don't care how careful you are, just do it. It takes 1/2 hour and my stuff works so much better and is soo much cleaner! I recommend Promega's little set of DNase with DNAse buffer and a stop solution. I use 1 ul 10x buffer, 2 ul DNase I, and .5 ul RNAse OUT (invitrogen) for no more than 2 ug RNA. It works wonderfully and only takes 1/2 hour. The DNase is EXTREMEMLY sensitive to any mechanical/physical stresses, so no vortexing etc, just pipette everything very gently!

2) Have you determined the optimal Mg concentration of the MMLV reverse transcriptase?
[I]We used to use the MMLV RT, and I switched the lab to superscript III by invitrogen. Basically it was the only thing in our supply freezer at 10pm when I needed it, but it works wonders. Mg is in the buffer, I use it according to manufacturers recommendations and it works wonders

3)What happens if you add more RNA template than recomended trying to improve cDNA yield? What is the concentration that you got?
I've tried this and I love it! ...but, you also have to remember to double your DNAse and I also let that go for 45 min instead. It worked well. Just reduce the amt of H20 in your cocktail when you mix up your RT!

Good luck, hope this helps!

I just want to say thanks for your precise information.