Hello Fellows,
My questions is regarding Qpcr. When I run Qpcr , I do replicates from the same sample to make sure data is consistent. House Keeping gene (L32) is always very consistent with me in all samples. However, some genes , in the same sample, show different CT values. For example: CT value of L32 will be 18.03, 18.02, 18.04. while CT value of gene X will be 30, 34, 36.
Is it bad primer issue, or machine issue, or hand issue, or others?
I appreciate your guidance.
Thank you.
Hello,
It seem that your target gene has very low expression levels. When you are at low number of cycles CT values a change 0.05 can mean tens or hundreds of thousand of copies of the gene in your sample. When you are after cycle 30 a change of 1 or 2 in CT means a very small change in number of copies of the gene in your sample. What is the CT value for your negative control. Do you use a standard curve? If you expect your gene to be properly expressed I would consider testing the efficiency of your primers.
Regards
To get more clarification, I'll suggest you to add your graph here so that people can take a better look at what's going on with your experiments.
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