I have done RNA isolation from a kidney cell line ( RIN 9.3), followed by reverse transcription using 1ug of RNA using iscript kit. But after qPCR, I found the positive control Ct value is 29 while the gene of interest only shows amplification at 40, irrespective of the amount of cDNA I add. Primer concentration is 300 nM and i tried two Tm (45 and 60).
The amplification of Ct at 40 regardless of your cDNA amount might suggest that non-specific product amplification which means there is no amplification of your gene of interest. Have you searched for general information regarding your gene of interest whether they are expressed normally in kidney cell? Any change of Ct observed from your positive control when you apply different amount?
Hello Jean, did you run a five points standard curve to determine the amount of cDNA you should use for your qPCR? You may try 10-fold dilution first for your standard curve, get the amount of cDNA you should use for your control and gene of interest to amplify below Ct 35..
The amplification of Ct at 40 regardless of your cDNA amount might suggest that non-specific product amplification which means there is no amplification of your gene of interest. Have you searched for general information regarding your gene of interest whether they are expressed normally in kidney cell? Any change of Ct observed from your positive control when you apply different amount?
I agree with Eric, go for it.
It is important to note that you might be targeting a low abundance transcript, and thus, it could be difficult to achieve reliable Cts (Below 30-32).
Plus, I would try an annealing temp gradient of 58-64 ºC (45ºC seems excessively low).
If you still have problems, you still could run a premplification on your samples, but this would require additional experiments to test that preamp is not affecting the results (ie, an additional standard curve).
Good luck
I just think whether you or your lab have any housekeeping gene primer (gapdh) for qPCR, you can try it to determine whether the samples (cDNA) is OK for the qPCR.
did you run regular PCR to see whether your primer is specific and efficient?
Hi Jean, if your melt curves are fine and as per your products and your GOIs are also not the low copy number ones, then you can troubleshoot it in the following way too, just make dilutions of your samples like 1:5, 1:10, 1:25. Perform one qPCR on all of these dilutions of samples and your original samples with new housekeeping genes (at least select 2 or 3 new HKG) , if still the same situation of delayed ct persists for all these new HKG, then there might be inhibitors present in your sample mainly related to extraction issue. If the Cts of diluted samples show some improvements than your original samples Cts so definitely you must revise your entire extraction properly and carefully & choose the most proper kit or protocol for this. By this way, you will also be able to know the best HKG for your experiment as well. Remember, that the Ct of HKG also declares the quality of your cDNA. Apart from impressive RNA RIN values the quality of cDNA also matters. There is something in your samples not allowing the proper amplification and these are no doubt some PCR inhibitors, a well known fact. In your case, their concentrations is bad enough and disturbing probably the polymerase during the RT step and / or in the qPCR phase to work efficiently.
The exact problem once happened to me and I sorted out easily by the way I explained above.
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