Hi everyone,
We usually use the Verso cDNA synthesis kit which includes something called "RT enhancer", which usually prevent carryover of gDNA into your cDNA samples. However we are out of this component but we do have DNAse I at our disposal.
The problem: the DNAse I kit says that you need to de-activate the enzyme at 65C (which is already pretty high for an RNA sample) in the presence of EDTA. Reverse transcriptase is Mg2+ dependent, which is sequestered by EDTA.
So how can I do a DNAse I treatment before RT-PCR without ending up with a bunch of EDTA in my reaction. I suppose I can just add extra MgCl2 solution to saturate the EDTA but the problem is that the exact components of the RT-PCR buffer from the Verso cDNA kit is unknown so I'm afraid of messing up the conditions for the reverse transcriptase, impairing efficient cDNA synthesis.
Thoughts? Thanks!
Benjamin Tak, you can try to precipitate RNA after DNAseI treatment and before reverse transcription http://cshprotocols.cshlp.org/content/2020/3/pdb.prot101717.full.pdf
Would be easier to filter the reaction via column based method to be sure that contaminants are removed which might assure or increase success of your following step.
qRT-PCR is notoriously sensitive to small variations in protocols. Anything you put into your cDNA synthesis (like excess magnesium) will be carried into your PCR, with a considerable risk of making your data incomparable with other data that you presumably already have. I would strongly suggest waiting until you have more of the component that you are missing (for best consistency) or else obtaining a kit designed for DNase treatment of RNA ahead of RT (eg Turbo DNase I) and then using it according to directions. You can probably also get away with an in-tube or on-column DNase I treatment followed by a column cleanup.
I was typing out an explanation about EDTA chelating in a 1:1 molar ratio and trying to calculate the exact amount needed based on what ions were in the DNase I reaction buffer - but it's qPCR and I wouldn't do it and I think you probably shouldn't either. Do it properly and have confidence in your results :)
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Thanks, Dan
Thank you to all the scientists for everything you do!
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