Question: qPCR: Huge variation in fold change of genes between biological replicates
0
gravatar for Ambika
13 months ago by
Ambika30
United States/Auburn/Auburn University
Ambika30 wrote:

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 foldchange • 781 views
ADD COMMENTlink modified 13 months ago by WouterDeCoster40k • written 13 months ago by Ambika30

It sounds like you have variable degradation of your samples.

ADD REPLYlink written 13 months ago by Devon Ryan91k

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

ADD REPLYlink written 13 months ago by Ambika30

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

ADD REPLYlink written 13 months ago by ATpoint19k

How are you determining fold-change? Are you using the ∆∆Ct method? If you don't normalise properly, you'll get meaningless results.

I presume that you've also checked DNA concentration?

I am using two reference genes EF and GAPDH and I also have variations in the Ct values of reference genes across various timepoints.

That's not surprising. During my PhD, I produced data that showed the inadequacy of housekeeper genes, and that was even using established primer pairs.

Generally, if you have processed your replicates equally then there should not be much differences. I also showed that PCR is a very precise method if using the same primers and samples.

ADD REPLYlink written 13 months ago by Kevin Blighe45k

@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.

ADD REPLYlink written 13 months ago by Ambika30

Is it circulating free DNA or FFPE-derived? Did you apply any 'whole genome amplificaion' like with phi-29?

ADD REPLYlink written 13 months ago by Kevin Blighe45k

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

ADD REPLYlink written 13 months ago by Ambika30
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