I've generated an enhancer knock-out in mice, which I then did qPCR of the gene it potentially regulates and seen it is 25% down-regulated in the homozygous knock-out. So I decided to do RNA Seq to analyze other genes, but in the RNA Seq data the gene isn's differentially expressed. I don't understand why, and which technique should I believe. The samples used for the qPCR and RNA Seq are not the same, but are the same genotype, same age, same tissue. The only technical difference is that I did Trizol extraction for the qPCR, but for the RNA Seq I did ARN column extraction.
Do a western blot, that should clear the difference. Variations created by different extraction methods can be substantial, and in RNA-seq you analyze all genes at the same time, while with RT-qPCR you only compare your housekeeping gene vs the knockout gene, hence your threshold will be different.
as Péter Gyarmati said the extraction method might introduce an important bias in the quantification results. How did you define "differentially expressed genes"? 25% of down expression will not pass the fold-change threshold in RNA-Seq analysis. In future experiments, it could be a good idea to add a spike in RNA-Seq samples to compare the results with RT-qPCR
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