Hallo experts,
I perform gene expression analysis using RT qPCR.
I compare two groups of maize kernels (group A and B). I have four pools per group and every pool consists of ten different maize kernels.
Now, I analysed the first gene and the CT values (of the specific gene) of the pools in group A are very different. Two pools have CT values around 19 and the other two pools have values around 21. The CT values of the housekeeping gene for these pools are the same. What is the reason for the difference between my pools in group A? Do you have any explanation?
How would you continue?
Thx,
Sina
Dear Sina, reason of the difference in Ct values of groups is the small differences in cDNA concentrations or pipetting errors. Did you equalize RNA or cDNA concentrations? before carrying out RT-PCR. Usually we compare each sample (target gene) with respect to housekeeping of the same sample.
Another reason may be expression differences for target gene in your maize samples.
Best regards.
Hello Sina,
First, I would like to check and make sure that your samples that belong to each pool did not get mixed accidentally
Second, I would like to check the steps because it might be technical reason
Third, your samples may have given different results, and in this case I would repeat the sampling and the extraction to make sure and be be confident.
Good luck
Thank you for the answers. I exclude RNA concentration differences as cause, because we measured housekeeping gene and target gene of all mentioned samples in one run in triplicate.
Rana you´re right, we have to repeat sampling and extraction to be sure.
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