Hi,
During QPCR, I ran non template controls and negative control RT reaction (no RT enzyme using random hexomers and oligodT primers) together with my normal cDNA samples in the same plate. After qPCR, I ran the PCR products on a gel to identify the bands for the product size. Note that all my primers are exon spanning.
The problem is that -RT samples showed amplification with expected size bands. The ct values were higher for -RT’s but not high enough to be negligible especially. If the ct value for my normal cDNA is 18, the Ct value for my -RT is 24. The reason for this cannot be genomic DNA contamination as the size of the bands are the same as the bands I got for the normal cDNA samples.
I still increased DNAse incubation time to account any effect of the gDNA but the ct values only increased by 2-3 values which still was not negligible.
I also run non template controls which did not show any ct values nor a band when I ran a gel. In this case I don’t think I have a contamination problem either.
When I ran the RNA only for some of them, I still had amplification with ct values similar to -RT controls.
I am worried that DNA polymerase is amplifying the RNA that is left in -RT samples. Do you have any other explanation for this? What can I do to prevent amplification in -RT samples? Any other thoughts?
Hi Ayse
- Did you blast verify your primers to make sure they only bind to the GOI CDS ?
- Also did you observe bands on agarose gels for the -RT controls ? If so try increasing the annealing temp by 1C and doing the same for actual qPCR reactions
Hi Asye,
1) You should use NAC (No Amplification Colntrol) - without DNA Taq Polymerase - to reveal that is there any genomic DNA contamination in your reverse transcription reaction.
2) If you haven't done, you should treat your RNA with DNase before reverse transcription.
If you exclude the above 1) and 2) isssues in the terms of gDNA contamination, then you can do a Primer-BLAST search (Primer-BLAST search (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)
to check your primers specificity to your GOI.
3) I suggest to do a touchdown PCR. If you can use PCR machine available with this feature. For the annealing temperature just set the highest temp for exmple 65-70 °C. Set the PCR to decrease the annealing temp by 0.5 °C after every cycle down to the calculated annealing temp of your primers. For example 55 °C (lowest temp). Run the PCR on this temp for the following calculated cycle number. For example: (total cycle number)-(numbers of decreasing temp cycles) = (remaining cycle number). E.g.: 40 cycles - 20 cycles = 20 cycles at 55 °C. This technique not as elegant as gradient PCR, but it can be usefull. Unf you won't know the ideal annealing temp like in grad PCR, but it will maximize the amount of the specific product. It's much more ellegant way to find the best annealing temp empirically to just run a gradient PCR if you available an machine with his feature.
Cheers,
Tamas
If this is not the first time you did this amplification, then the most likely explanation is contamination from the PCR products you have made before. PCR products make the perfect contaminant and it takes a great deal of care to avoid this. It is so easy to get minor contamination in your pipetting devices. Thus, we routinely have totally different sets of pipettes for setting up PCR reactions and for analyzing the products.
Your RNA contains genomic DNA, DNAse I treatment is necessary:
https://www.thermofisher.com/in/en/home/references/ambion-tech-support/rna-isolation/tech-notes/a-new-method-to-remove-dna.html
Thank you for the answers, really appreciated
@Laurence, @Tamas and @Ruslan, yes I did blast verify my primers and they are specific to the GOI and yes I did see bands on agarose gel for -RTs. I did gradient annealing and optimised the best annealing temp for all my primers by confirming single bands on agarose gel. But I did not try gradient anneal for my -RTs. I could try that.
If it was a gDNA contamination shouldnt the product size be much larger (~1000-1500 base pairs) as my primers are exon spanning. Instead I get the expected size of band (~200-400 base pairs) that corresponds to my GOI.
@Prescott Deininger, if it was contamination issue shouldnt I have a problem with my non template controls as well? But my NTC pcr product comes out clean with no ct values and no bands on agarose gel.
Note that I have updated my question with further information.
Regards,
Ayse
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