Hello everyone,
I am conducting the qPCR to check the gene expression. In spite of high reaction efficiency of the housekeeping gene, my gene of interest amplifies very low as the ct value is higher than 30. I have tried many ways to troubleshoot this out by increasing the concentration of the primer, the concentration of the cDNA, or adjusting the annealing temperature of the thermal cycling condition, but the result showed no difference.
I think it might be due to the primer used for the gene of interest because the housekeeping gene shows very good result (the picture of standard curve is attached)
The primer that is used for the gene of interest span the exon-intron junction, the length is 20bp for both forward and reverse primer, Tm of forward primer is 61.1 and reverse primer is 59.1, and the length of the product is 256bp.
I also attach the picture of amplification plot. A and B line are plotted from the target gene, C and D are plotted from the housekeeping gene (I used beta actin).
Does anyone has any idea to troubleshoot this problem?
Thanks in advance.
hi see this link https://www.thermofisher.com/.../pcr/real-time-pcr/qpcr.
Measure the efficiency of your gene of interest primers. If it's between 95 & 105%, then you just have a gene of interest with a low level of expression.
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