Discrepancies between deep sequencing and qRT-PCR results

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Mojca Strazisar

according to http://www.ncbi.nlm.nih.gov/pubmed/20815927 it is based on library prep. although the whole research strategy in the paper is for me somewhat questionable .

Check this one too http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287988/?tool=pubmed

BTW qpcr -did you do relative quantification? If so your result strongly depends on the controls used for normalization.

Hope it helps a little.

Jun Xie

It could be qRT-PCR detect only one isoform of your miRNA, but deep sequencing data shows all.

Vladimir Ivanov Vladimirov

We all might be of greater help if you tell us a bit more about your experiment. What samples the RNA was isolated (cells or tissue)? Waht's the quality? Usually RNA from fresh tissue or cells is of much higher quality compared to paraffin blocks or postmortem (e.g. Brain) tissue. Did you do miRNA-Seq or total RNA-Seq? That will affect the library preparation, but more importantly the normalization approaches, which for RNA-Seq are still rudimentary. I'm sure you've thought about it, but what statistical approaches did you use to analyze your data? Did you account for potential confounding effects in the RNA-Seq and qPCR analyses? how did you control for multiple testing? how many of the significant results, what fold change, did you attempt to verify?

Niti Jadeja

i agree with Jun Xie, that probably might be the case, but could u share ur rna isolation steps?

Muhasin Asaf

The experiment was miRNA expression profiling during avian influenza infection. There were two groups, H5N1 infected and SPF birds. The sample for the experiment was lung tissue obtained after sacrificing the bird and was stored in RNAlater. The results obtained by deep sequencing has identified differentially expressed miRNAs as upregulated and downregulated. Then we have performed qRT-PCR experiment. RNA was isolated using mirVana miRNA Isolation Kit. NCode miRNA First-Strand cDNA Synthesis and qRT-PCR Kits was used for the experiment. The discrepancy occurred only to the upregulated miRNAs while for the downregulated miRNAs the deep sequencing and the qRT-PCR are tallying.