Real time PCR in the presence of heme - (Jun/03/2011 )
Hi all, i'm having problems with extracting RNA from samples that I know are contaminated with heme.
I've been told that PCR can be inhibited when in presence of heme, and would like opinions on how I could overcome this or if you have ever faced similar problems?
Just so you have an idea I'm quantifying parasites in mice livers both by RtqPCR and Luminescence and the values of luminescence are always higher, which I think it maybe because my RNA samples are contaminated with heme and hence not giving the same results...
Thanks in advance!
How do you isolate your RNA? Most RNA kits will clean it up so there is no heme contamination. And absolute quantifying always relies on standards, are your standards for both methods trustworthy?
Hi Trof, I'm using a kit from invitrogen that is usually used in our lab for liver RNA extraction. I'm concerned that heme being such a small molecule could still get to the final RNA eluate and inhibit the PCR reaction. I don't use standards, but normalize the CT values of my infected mice with the CT values of uninfected mice. problem is that I expect that the infected group will have a higher heme content than the uninfected group (meaning i could potentially have more inhibition in one than the other)
Is that Invitrogen kit a column kit? Because from what I know about column kits, they work a principle of catching nucleid acids on the column and washing everything out with a set of washes, so heme being small would be first to go.