After performing qPCR on intestinal tissue collected from animals, I have high variability in Ct (difference of 5-7 Ct cycles) values within treatment groups (between biological replicates - 10 samples per treatment group).
My technical replicates (same sample tested in duplicate on the same plate) consistently have identical Ct values. However, between biological replicates there is major variation.
Is this normal? Within each treatment group (biological replicates) there are different genders (both male and female), housing conditions etc.
Is the difference in Ct values within biological replicates due to natural biological variation or is there some other factor involved?
Does anyone have any experience carrying out qPCR on in vivo samples?
Is your PCR data normalised to a housekeeping gene? If so, which one?. It's possible the variation is due to a housekeeping gene that may not be the most appropriate.
Hello,
was the extraction of RNA performed with the same reagents, at the same time? Also, have you checked the purity of the isolated RNA?
As Mr. Wyn Jone stated, you could try different housekeeping genes, however, I believe that the differences in the biological samples (for ex. gender) may also result in high variations between the samples.
The qPCR dats is normalised to a reference gene using the 2–∆∆Ct method. I have tried RPLP0, RPLP1 and B2M as reference genes and all displayed variation between biological replicates.
RNA extractions were performed using the same reagents from the RNeasy Mini Kit from Qiagen followed by cDNA synthesis using the SensiFAST™ cDNA Synthesis Kit from Meridian Bioscience.