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RNA isolation for real-time RT-PCR


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4 replies to this topic

#1 pcrstarter

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Posted 25 June 2009 - 12:24 PM

After RNA isolation by using Tri reagens (and chloroform,...), I did a DNAse digestion with a kit from Invitrogen. I added EDTA, BUT I did not the DNAse inactivation step at 65C! I thought the thermocycler was at 65C, but I forgot to press the start button! So the actual temperature was room temperature.
Without noticing, I continued measuring RNA and then diluting with water. I stored the RNA samples at -20C.

Can I proceed the RT-PCR reaction with these samples if the DNAse is not inactivated at 65C?

#2 pcrman

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Posted 25 June 2009 - 06:11 PM

I think you need to inactivate DNase before RT reaction.

#3 Carlton H

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Posted 25 June 2009 - 07:21 PM

I'm about 80% certain that there are companies that make DNAse-inactivating reagents that do not require heating, but I don't know what they are. ... May be worth a bit of an internet search and if it can salvage the samples, though.

Cheers,
-Carlton
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#4 Curtis

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Posted 26 June 2009 - 12:21 PM

I agree with Carlton, but i'm still a little in doubt for my own case;

I have this cDNA synthesis kit from Qiagen which has a DNase vial in it called gDNA. The protocol does not require any heating or chemical-inactivation. I just add this gDNA to my RNA sample for 2 min and then put on ice and add the rest of the RT reagents. I was also surprised when I first read the protocol, because in standard protocols we normally need to add stopping reagents that contain EDTA or something.

So I'm also concerned if this gDNA really removes all DNA inside my RNA sample, otherwise my PCR is giving me me false result!

#5 molgen

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Posted 08 July 2009 - 07:21 AM

Don't you start your qPCR with 5-8 min at 95C?
In my mind if the inactivation is at 65C you shouldn't have a problem.




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