Cleanign up RNA isolation - (Dec/19/2007 )
HI guys!
I have been reading about RNA treatment with RNAse free-DNAse free after extracting it with for example TRIzol. My doubt is: what is that for? I extract viral RNA and do RT-PCR with it without any treatment and have no problems, does it influence in the amplification efficiency? Thank you
-debokuki-
QUOTE (debokuki @ Dec 19 2007, 08:23 AM)
HI guys!
I have been reading about RNA treatment with RNAse free-DNAse free after extracting it with for example TRIzol. My doubt is: what is that for? I extract viral RNA and do RT-PCR with it without any treatment and have no problems, does it influence in the amplification efficiency? Thank you
I have been reading about RNA treatment with RNAse free-DNAse free after extracting it with for example TRIzol. My doubt is: what is that for? I extract viral RNA and do RT-PCR with it without any treatment and have no problems, does it influence in the amplification efficiency? Thank you
For tricky RTPCR it often helps to remove any left over genomic DNA that might cause misinterpretation of results. This is especially the case if your gene of interest doesn't contain introns because then you cannot design primers that would distinguish cDNA from genomic DNA. Being that you're extracting viral RNA, I'm not sure if these same concerns would apply to you.
If you haven't already done so, you should probably set up a mock RT reaction to test if you need to be concerned about DNA contamination. Set up your reverse transcription reaction as you normally would - only don't add the reverse transcriptase. Perform PCR as normal. If you don't get any product, then great - you don't have to worry about it. If you do get a product then you should perform the DNase treatment.
-smu2-
exactly what smu2 said.
V
-vetticus3-
Hey!! Thank you very much! I will follow your advice.
-debokuki-