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

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

qpcr fold-change
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updated by Ram Ram • written by AP • AP

It sounds like you have variable degradation of your samples.

• 1 views • link
written by Devon Ryan Devon Ryan

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

• 1 views • link
written by AP • AP

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

• 1 views • link
written by ATpoint ATpoint

@Kevin Yes I am using ∆∆Ct method to determine the fold change. I collected all my biological samples the same day in order to minimize the variation, and of course the treatments and conditions are same. Even my technical replicates look fine. So, I am having hard time figuring out what is the actual problem.

• 1 views • link
written by AP • AP

@Kevin I am just using cDNA to amplify the genes. I did not apply any whole genome amplification

• 1 views • link
written by AP • AP

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