Focus a lot of energy on finding a good DNase treatment of your RNA extracts. TURBO DNase has been reported to be 300X more kinetically active than wild-type DNAse I - so perfecting the use of that may help a great deal since you don't have the option of being able to span genomic exon-intron junctions by primer design. Prokaryotes (procaryotes); e.g., bacteria and archae, lack introns, so, with RT-PCR and/or RT-qPCR, you always run the risk of amplifying gDNA if you haven't successfully digested it away beforehand.
A good formulation of TURBO DNase treatment:
10 uL 10X TURBO Buffer
70 uL sample RNA
20 uL TURBO DNAse enzyme (an excess - which is OK)
Incubate 30 minutes at 37C.
Then, at room temp:
Add 10 uL Inactivation reagent, vortex for 15 seconds off and on for 2 minutes,
then spin at 10,000 x g for 3 minutes (not 1 minute as suggested), and remove 80 uL of the supernatant - leaving the inactivation reagent and some sample behind. Be absolutely careful to not grab any inactivation reagent when you take the 80 uL of supernatant.
Dilute the 80 uL then 1:10 with 710 uL of nuclease-free water and 10 uL of RNaseOUT (Invitrogen). You now have 800 uL of very well-DNased RNA sample.
This is the starting concentration of your RNA.
At this point, do a dilution series and find the optimal dilution range of the sample.
After using this method for years, my gDNA signal is always either non-existent or at least 13 cycles later than the genuine RNA/cDNA signal; thus bearing negligible consequence on quantification.
Thank you so much sir. I have been doing DNase I treatment and I am getting results.I will try TURBO and hopefully the quality of results will increase.
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