Reference genes from RNAseq data for qPCR assays

Question

[AP]

Hello everyone,

I am trying to validate my RNAseq results through qPCR but I am having trouble in finding the stable reference gene. Is there any way I can use my RNA seq data to find the stably expressed genes?

Please help.

Thank you,

Ambika

Answer 1

[h.mon]

There are some papers that use RNAseq to select refence genes, e.g.:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357963/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342825/

[AP]

h.mon

As far as I know they use cq or Ct values of the selected genes in order to find the best stable genes using some programs. My question here is, how can I pick those genes and on the basis of what? Can I use my LFC expression data ? Once I pick some genes then I think I can compare their cq values to find the best ones.

[h.mon]

From Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem:

In order to rank the predicted 34,725 tomato genes (ITAG 2.4) based on their transcript level stability, we calculated the variation coefficient (VC) using all the RPKMs (reads per kilobase of transcript per million mapped reads) determined in each experimental condition (37 different conditions, Supplementary Table S2) using the biological replicate information individually. This accounted for 110 total values. The lower the VC is, the more stable the expression of the gene is across all the conditions. In this way we selected 9 genes with the lowest VC for analysis, none of which had been used previously as tomato reference genes in RT-qPCR assays (Supplementary Table S2).