My "no template" controls are showing products that are identical to their genes. I did bax and p53 qPCR and noticed that bax amplification was almost identical to bax no template control; p53, the same. Their Cq values were also identical. Their melting curves also looked the same. A single peak was shown on their melting curve. Is the problem with my primers? What other issues could cause this? DNA contamination of reagents?
Have you sequenced the product and/or run it on a gel? This could only be contamination of your RNA or amplification of gDNA.
Does your primers span an junction Exon / Exon or does your primers are on two different exons ? Do you perform, after or during RNA purification, a DNase treatment ? If not, it might be a contamination by gDNA.
Hi Ahmed and Oliver, thanks for your responses. My concern is why NT control should have same Cq, melting peak, and similar efficiency (judging from the slope of their amp. curves). I do not think a "random" contamination could cause this. Oliver, I am running a 2% agarose gel to verify an approximate of the product sizes. Also, if i had "some" gDNA contam., will the curves look that identical?. Ahmed, if my primers are not single-target specific, why do I get one melting peak?
I guess that with NTC you mean that you are putting in the reaction just water instead of cDNA... so RNA and gDNA should not be your problem. But you clearly have a contamination in any of your reagents. It could be a cDNA contamination or, if in the lab you are working with these genes (bax and p53), you might have plenty of plasmids containing the CDS of these genes, and this might be your contamination, mostly if you don't use dedicated pipettes, plugged tips and a dedicated hood/bench for your RT-qPCRs. I agree with Ahmed that if your cycle number is high, around 35, you could get Ct values in NTC close to your samples' even with a small contamination in your primers or buffers or dNTPs or water. But if you have plasmid DNA contamination in any of your reagents you can easily get much lower Ct values even with a low contamination.
I suggest that you throw away all your reagents where you dipped a tip in, clean up thoroughly your pipettes in 1M NaOH and restart with the clean tools and plugged tips by extracting the RNA, doing your RT and finally the qPCR.
Good luck!
What do you mean by "not single target specific"? Your QPCR primers have to be specific to a single target. I would throw out all the reagents and start afresh. Make fresh primer stocks and get fresh aliquots of your reagents.Try setting up a plate with just "no template control" and no RT control, see what you get.
1) your dnase treatment failed
2) contamination
3) primer produce product by self hybridization
As simple as I could, I am describing the possible things gone wrong....
Thanks y'all for your help. Yes, I do DNase treatment during my RNA extraction. I am running 40 cycles. I ran a 2% agarose gel and realized that they were all primer dimers (Yes Georgios). They were not my target. So so far, I think my reagents are not that bad.I am currently optimizing annealing temperatures.
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