Cycle thresholds are about 25-29, and the product bands are weak on agarose gel electrophoresis.
What extent of ethanol contamination?
If you do a dilution series of the RNA in question, and the Cq values improve - then you know the ethanol is waging an influence. A minor amount of ethanol shouldn't hurt things - but your A260/A280 and/or A230 measurements will be a little strange, unless you zero the spec. or NanoDrop with a similarly-ethanol-contaminated blank.
Ample dilution of the samples before RT-qPCR could get you out of this mess unless your targets are low in abundance in the first place (biologically); you don't want to dilute yourself into oblivion of course...
while doing the RNA extraction process I had no knowledge about the inhibitory effect of ethanol on amplification so the amount of contamination is realy high(20 macro litre of ethanol in an RNA extract with 70 macro litre volume).
I have done a dilution serie of cDNA in order to gain the efficiency of primers. but strangely, the Ct of 1/100000 concentration is the same as 1/10 concentration. my hypothesis is when I dilute the cDNA, this inhibitor is diluted simultaneously so the inhibitory effect of ethanol is decreasing in 1/100000 concentration versus 1/10 consentration, and this results in the same Ct and the same amplification.
Perhaps then the ethanol in the RNA extracts (~28.6% ethanol) is inhibitory to the reverse transcriptase during cDNA synthesis and your cDNA is what has suffered in some way. If the Cq value is the same no matter the dilution, it sounds as if you are having very severe primer dimer issues - which would get more pronounced the more you dilute your template cDNA. This is a very strange situation otherwise. I am not sure what ethanol does to the effective Tm of random hexamers and/or olgio d[T]n priming during reverse transcription. Ethanol forms an azeotrope with water, and that azeotrope has properties distinct from either water or ethanol individually. Ethanol can also affect the sphere of hydration of the RT enzyme; affecting its activity. My theory is that you got little cDNA from your reactions, and all you are amplifying are primer-dimers (yes even as low as 25-29 Cq) and/or some other freak thermodynamically-possible product that stays constant regardless of cDNA dilution due to interaction of primers with themselves in conjunction with the left over unincorporated randomers or oligo dTs you may have used for cDNA synthesis. Are you performing SYBR Green based qPCR? If so - what does the melt curve look like? Is it a classic specific product peak at a reasonable Tm?, or a lower rounded hill shape in a lower temp region indicative of primer-dimer?
yes I use SYBR green based qPCR. but the problem is not related to primer dimers because of two reason: 1) gel electrophoresis of qPCR products show bands equal to the target gene (280 bp) but not primer dimers.
2) the melt curve peaks have no signs of primer dimer existence
I too once had the experience of no dilution effect on Cq values but it was with a one-step SYBR Green system wherein the RT and Taq enzymes were both in the mastermix. I could never come up with an explanation for this other than that the BAL (bronchoalveolar lavage fluid) from which the RNAs were originally isolated contained some carry-over contaminant that caused this. I never identified the problem (though someone else isolated the RNA and ran the RT-qPCR). Your situation is indeed puzzling, especially with your gel and melt curve analyses showing no primer dimer.
An interesting publication mentioning the inhibitory effect of ethanol states the following:
"Perhaps a more thorough approach would be to assess the relevant PCR reactions for inhibition using a range of concentrations of several well known inhibitors such as heparin and ethanol in addition to EDTA. Ideally, nucleic acid extracts from the sample types of interest (assuming they are known not to contain the PCR target) should also be tested for their inhibitory potential with the relevant PCR reactions but this may not always be feasible..."
This paper is at:
http://www.biomedcentral.com/1756-0500/1/70
Perhaps this publication holds some key insights for you?
thanks for the paper jack, I will inhibit ethanol contamination in my new extraction processes
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