qPCR: Huge variation in fold change of genes between biological replicates

Question

[AP]

Hello everyone,

I am trying to validate my RNAseq data by doing qpcr for which I am looking at the fold change of few genes across various timepoints of treatment conditions. I am getting huge amount of variation (in thousand folds ) in my biological replicates. I thought may be it is due to genomic DNA contamination so thats why I repeated all my experiment and did DNase treatment twice but still I am having such variations. I am using two reference genes EF and GAPDH and I also have variations in the Ct values of reference genes across various timepoints. I will really appreciate it if you could suggest me with possible problems and solutions.

Thank you,
Ambika

[Devon Ryan]

It sounds like you have variable degradation of your samples.

[AP]

i already checked the degradation by running gel on my RNA samples before and after DNAse treatment. They look fine.

[ATpoint]

As this is purely wet-lab, I may redirect you to Stackexchange to ask this question.