I have performed RNA extraction of blood sample using TRIzol method. I ran RNA samples on non-denaturing gel (1.5%) that showed extreme DNA contamination and degraded RNA band. Please suggest me how should I overcome this issue.
Did you make any effort to denature your RNA before running it on the gel? RNA is mostly ribosomes, and these have a lot of secondary structure: just running these on a standard DNA gel will not usually be particularly informative.
You don't need to run denaturing gels, either: you can just denature your RNA first: mix the RNA samples with 6x loading buffer and formamide, aiming to get the formamide % as high as possible, ideally 60-75% (1ul RNA, 1ul 6x loading buffer, 4ul formamide, for example).
Heat these samples at 65 degrees for 5 mins, then crash cool on ice. This denatures the secondary structures and then the ice + formamide prevents them reforming.
Now run this on a gel as normal. Use an appropriate ssRNA ladder, obviously.
Longer term concerns: how are you isolating your RNA? Whole blood or PBMCs only, or...what? If the former, you will have a ton of erythrocyte specific RNA but should not have anything like as much DNA contamination, on account of RBCs not having any DNA.
If the latter, then you might get gDNA carryover, but it shouldn't be significant.
One tip is to be more stringent when collecting the aqueous phase immediately after phase separation (material close to the interphase will be more likely to contain gDNA).
Another tip is to just...DNAse treat the RNA after isolation. Or, if this is just for qPCR, to design primers to span introns, such that only cDNA amplicons are valid targets.
Hey John,
1) I didn't do denaturation of RNA samples.
2) I used whole blood.
I have to perform RT-qPCR, so even if I get RNA bands I'm worried about DNA contamination
Right, so...again: whole blood won't have a lot of DNA in it. Most of your RNA will be from erythrocytes, but maybe that's what you want.
And again: DNA contamination is only a problem if you make it a problem. Design primers to span introns (this means gDNA won't be amplified), but even if you can't do this, you can just run -RT controls and empirically demonstrate that gDNA contamination (if present) is sufficiently low so as to leave your conclusions unaffected.
Basically, if you run a qPCR with cDNA samples prepared without reverse transcriptase (such that all that can be amplified is genomic DNA contamination), and all your Cq values are ~6 cycles higher than they are when using proper cDNA, then...you really don't need to worry: this corresponds to a 64-fold difference, i.e. genomic DNA represents
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