I am trying to check gene expression in a few cell lines, but the problem is that every time the results of Real-time PCR gives error 1 and 2, which means that in my sample cDNA concentration is low and some sample gives multiple peaks. This never happens with control primer. I increased the cDNA concentration in dilution but the problem still exists. The results show overexpression of the gene in that cell line, but I am not sure whether the results are correct or not. Is there any problem with my primer?
Madiha,
I agree with all of the above comments but question whether it is worthwhile to troubleshoot your current primer set or just design new primers. As stated or inferred in the other responses, make sure that the primers span an intron and, when blasted, do not have similar homology to other transcripts. In addition, I wonder if the transcript in question is expressed at very low levels. You may want to refrain from diluting your cDNA to much prior to performing QPCR. Alternatively, you may want to think about using taqman probes to give you increased specificity and sensitivity. Best of luck with your research.
Hi Madiha, I fear the problem is in your primers, they either recognise more than one target or do strange things when the target is genuinely low.
Do you see higher Ct numbers for the target gene (compared to reference gene)
Have you tried a positive control with these primers. For example a cloned fragment of the cDNA (spliced as you expect) or even genomic DNA if the primers don't span an intron. You can try with positive control alone, or with a mix along side that has the positive fragment and all other potential targets from the cDNA.
Good luck
Hi Madiha,
I think that the problem might be in primers lenght; you should try with some shorter primers, increasing annealing temperature to assure more specificity.
Hi
Blast both your primers and check the results. If both primers target more than one gene in the same organism, are unsuitable. As said before, your problem is most likely the design of the gene specific primers.
Madiha,
I agree with all of the above comments but question whether it is worthwhile to troubleshoot your current primer set or just design new primers. As stated or inferred in the other responses, make sure that the primers span an intron and, when blasted, do not have similar homology to other transcripts. In addition, I wonder if the transcript in question is expressed at very low levels. You may want to refrain from diluting your cDNA to much prior to performing QPCR. Alternatively, you may want to think about using taqman probes to give you increased specificity and sensitivity. Best of luck with your research.
Hi Madiha
1) Check the specificity of your primers on Primer Blast programs at NCBI website...if you have designed for the first time..to see undesirable interactions with other amplicons or within and between the primers too...
2) Optimize the PCR conditions on a regular PCR first...if everything seems fine then shift to RT-PCR
3) carefully analyze the melting curve...it would be nice and informative if you
can load the photograph of melt curve..
bye
Jonathon Winnay makes excellent remarks. Additionally: if your primers span an intron, check its size. If your intron is small (~ 100 bp) and your gene is lowly expressed, a good quality Taq-polymerase will have no problem amplifying traces of genomic DNA. The first time you run an RT-qPCR reaction with new primers, you should verify on gel if your product has the expected length, and if multiple bands are present, if there is a gDNA comtamination.
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