Genomic DNA contamination of RNA - (Jun/10/2009 )
The first thing I would try is to skip the RNA recovery after each digestion -- just digest it three times, adding DNAse as appropriate to the volume each time, and recover the RNA once after you're done with the third digestion (or do one digestion with three times the amount of DNAse?).
If you still lose a lot of RNA with lithium precipitation, consider adding a carrier molecule like oyster shell glycogen to improve recovery, or try column recovery.
I think our Pippin Prep instrument could, in theory, cleanly extract RNA from genomic DNA. Will check with our development group about a making special precast cassette for this purpose. If we build a cassette for this purpose, is anyone interested in beta testing?
What are the relative sizes of the RNA and DNA in question? Easy to parse electrophoretically?
I use Qiagen RNeasy kit with on-column DNA digestion step and it removes all DNA only in 20% of the cases. This is why I follow up with TurboDNase kit with EDTA addition and heat deactivation. The DNA is removed but I encounter another problem.. After cDNA synthesis the PCR doesn't work! I checked up all possibilities (RNA degradation, cDNA synthesis fail, PCR products etc), everything leads to Turbo DNase buffer..
To test it, I used DNA template that works and mixed it with one of the cDNA samples that does not give results, also I added aliquot of DNase buffer to my DNA sample and included positive control (just the DNA). After standard PCR, only positive control gave good band; the mix between DNA and cDNA showed very faint band and third sample failed completely! Did anyone have similar problems?
I tried to purify my cDNA on a column, precipitate, add betaine, caseine, BSA, adjust Mg2+ concentration, dilute, add 1% Triton-X.. all for nothing.
Only 10x dilution and addition of 1% Triton gave me very faint band.. and in 2 cases (out of 40) precipitation restored template activity.
After column purification or precipitation my samples are clean (perfect spectra on NanoDrop) and of ca. 30 ng/ul concentration. Gels of RNA and cDNA look good too.
I am desperate.
The straightest way to differentiate amount of DNA from RNA is to run RT with and with out reverse transcriptase in it, the delta Ct between them is the indication of the RNA amount, while Ct from RT free indicates the DNA amount. RNAMono assay from Metammune.com exclusively amplifies RNA from DNA background, go check their design, you can design your own assay their way.
You can never get rid of all genomic DNA contamination. One way to measure it if you are anal retentive about it is to have a pair of primers that both lie in the same exon - do a real time experiment with/without RT in the cDNA synthesis step - theoretically you shuold get no signal from the no-RT control. The delta Ct between the no-RT and +RT will tell you the % genomic DNA (of course, there is variation in the RT efficiency), but you'll still get the idea.