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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

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  • $\begingroup$ How do your technical replicates look? $\endgroup$ – Rover Eye Jun 20 '18 at 16:21
  • $\begingroup$ To clarify, the variation you're seeing in the qPCR isn't being seen in the RNAseq? Do the values correlate? How was the qPCR data normalized? $\endgroup$ – CKM Jun 20 '18 at 16:46
  • $\begingroup$ How are you aliquoting DNA samples into your reaction mix? You can get alot of error just from errors in the pipette. I fine a ECHO liquid dispenser makes all your troubles go away (provided your can find one somewhere) GAPDH is sensitive to metaoblic state (ie oxgyen and glucose levels.) Are you using column purification of RNA with DNAse treatment? $\endgroup$ – JayCkat Jun 20 '18 at 17:45
  • $\begingroup$ @RoverEye my technical replicates look good, they don't have such variation. $\endgroup$ – ambika Jun 20 '18 at 18:12
  • $\begingroup$ @CKM I made PCA plot to compare my replicates for my RNAseq data. I had 4 replicates for RNAseq in which there was clustering of 1 and 2 together ; and 3 and 4 together. But that might be due to environmental variation since I did that in different time. In qPCR I am seeing the expression of those genes but the fold expression between biological replicates do not match. They have the same trend though. I am having 1000 folds difference in some genes $\endgroup$ – ambika Jun 20 '18 at 18:19

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