RNA isolation for real-time RT-PCR
Posted 25 June 2009 - 12:24 PM
Without noticing, I continued measuring RNA and then diluting with water. I stored the RNA samples at -20°C.
Can I proceed the RT-PCR reaction with these samples if the DNAse is not inactivated at 65°C?
Posted 25 June 2009 - 07:21 PM
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Posted 26 June 2009 - 12:21 PM
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!
Posted 08 July 2009 - 07:21 AM
In my mind if the inactivation is at 65°C you shouldn't have a problem.