So I want to DNase treat my RNA and inactivate the DNase. Then later synthesize cDNA and do qPCR
The DNase inactivation is often done via heating 10-20 min at 65-75C.
However, cations can mediate RNA cleavage at high temperatures.
So, to protect the RNA, EDTA is added prior to the heat inactivation of DNase.
However, the EDTA can interfere with downstream applications like cDNA synthesis.
So can I just add an equimolar amount of MgCl2 after the heat inactivation? Will that cancel out the EDTA?
Like such:
1. Add DNase to RNA sample and incubate
2. Add 1 uL of 0.5 M EDTA
3. Heat inactivate
4. Add 1 uL of 0.5 M MgCl2
5. Add primers and reverse transcriptase without purifying
Will X moles of MgCl2 cancel out X moles of EDTA?
- Yes heat plus Cations as you say can mediate RNA cleavage
- This is seen when you run out RNA and examine subunits
- In principle there is no reason why you cannot do what you suggest
- However, moist people do not so that I notice your time for inactivation is very long, even considering the lower temperatures
- If it were me I would (and have) used 5-10 min inactivation at 60C-70C; That should be fine
- In truth, a lot of people who perform RT PCR tend not to worry about this type of hydrolysis in terms of RNA degradation as a small amount of degradation is still conducive to efficient and accurate RT PCR
- Thus, add in your EDTA to kill the reaction and either omit the heat inactivation or perform inactivation for 5 min at 70C
- The run 100 to 200ng of total RNA on an agarose gel and providing you can see distinct subunits even with some smearing between them that will be fine for cDNA synthesis and PCR
Thank you. I will check the RNA integrity post-DNase treatment and likely try less severe inactivation conditions. I would worry about skipping the heat incubation entirely as I would imagine the DNase could become active when the product is diluted into the reverse transcription reaction.
My main concern is the effects on qPCR and reverse transcription.
Could the presence of high concentrations of EDTA+MgCl2 potentially interfere with the reverse transcription and subsequent qPCR? And is an equi-molar quantity of both the correct choice?
I am hoping they simply cancel each other and do not affect anything -- as they bind 1:1 as I understand.
I'm sure the safest thing is phenol-chloroform extraction/column purification to get rid of the DNase, but that can be quite a burden for large numbers of samples, plus there will surely be a fair bit of product lost.
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