I have extracted total RNA from Lactobacilli and treated the samples with DNase I. When checking the -RT on qPCR, there was always gDNA contamination (appeared at cycle 33 or 35, automatic threshold) although the DNase incubation time was extended (normally 20 min, I extended 40, 50, 60, 70, and 80 min) either the enzyme concentration was increased (20%). Somebody said that it is hard to remove gDNA totally but it is still okay if the signal from gDNA does not appear before 30 Ct. I wonder if it is true or not, and would like to ask everyone for opinions about this issue.
The image shows up the result of undiluted -RT RNA sample (with and without DNase treatment). The NTC shows no amplification. With this result, can I proceed with my qPCR for relative quantification, or it need further DNase treatment?
Short answer: yes, you can proceed.
The fact you're bothering to check puts you ahead of many investigators, in all honesty.
The relevance of gDNA contamination is strictly-speaking a relative issue rather than a constant one: if you're measuring a rare transcript, gDNA contamination might matter, whereas if you're measuring an abundant transcript, it really doesn't.
My rule of thumb is to ensure you have at least 6 cycles of difference between your measured GOI expression and your -RT gDNA Cq values. 6 cycles corresponds to a 64-fold difference, i.e. your gDNA contamination, if present, is only contributing 1.5% of your measured values: substantially below the sort of typical well-to-well variation you'd get simply from minor pipetting errors.
So if your -RT Cqs are all 33-35, and your GOI Cqs are all lower than ~27, you're fine.
Hello Tran
If you study this paper: doi: 10.1373/clinchem.2008.112797, then you do not think of skipping DNase treatment, no matter low or high degrees of contamination. It is a "must do" in qPCR experiments.
Thank you very much for your advice John Hildyard and Ali Javadmanesh
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