RNA PCR - RNA PCR (Nov/24/2008 )
Hi, last week I asked for hekp on my RNA extraction and purification. I am still having problems to have my RNA clean of genomic DNA. Today I would like to ask you something else. What do you do to confirm the presence of genomic DNA in your RNA sample????? So far what I was doing was set up a normal PCR but using RNA sample. Normally I got the same band than using DNA, therefore I conclude that there is still genomic DNA in my sample. Is that correct????
Yes, that is correct.
If you are having real problems, try designing primers that span intron/exon boundaries. They should amplify only from cDNA not genomic.
it depends on where your primers bind. If they bind to the same exon, you will get the same product size for DNA and RNA. To test for DNA in your RNA prep you could design pirmers that bind to two different exons. So amplification of genomic DNA will result in a larger PCR product because of the interspersed intron.
Unless you use oligo-T purification, all RNA extraction methods almost always have genomic DNA contamination. DNaseI digest works. You can and should always do a no-RT control to ensure complete DNaseI digest. That is, set up an RT reaction extactly as your regular RT-PCR but without adding the RT. If you get the amplicon, you have gDNA contamination.
I understand, but I set up a normal reaction, using RNA rather than cDNA as a template and what I got is the same than using my cDNA and genomic DNA. I think that means that genomic DNA is present. I have been told that maybe I should treat my RNA using restriction enzyme previous to DNAse and so it will be easier for DNAse to digest any trace of genomic DNA in my RNA sample. Is that possible????
Thank you for your answer.