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How to find stable housekeeping genes from RNA seq data?

I'm looking to validate key DEGs using qPCR after performing DGE analysis using RNAseq data. Instead of selecting canonical housekeeping genes as a reference I would like to use my RNAseq data to determine the most stable genes. How are people doing this? I'm looking at Normfinder, but the macros neither works for the Mac version of excel and the R package doesn't seem to work for me either. Importantly, I want to make an evidence based decision before obtaining qPCR data.

Please let me know!

Marina

rnaseq hkg
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updated by h.mon h.mon • written by marina.wakid • marina.wakid

Since you have already done DE analysis, you can select any gene(s) that is showing reasonably constant expression in your dataset?

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written by GenoMax GenoMax

To be systematic about 'constant expression' per gene, would calculating variance per gene be good enough? (for genes that are DEGs of course).

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written by marina.wakid • marina.wakid

On the contrary, you are looking for genes that aren't differentially expressed.

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written by h.mon h.mon

Sorry! I meant to say for genes that AREN'T DEGs. My mistake!

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written by marina.wakid • marina.wakid

1 answer

There is a lot of references about the subject. E.g., two practical examples:

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

Systematic identification and validation of the reference genes from 60 RNA-Seq libraries in the scallop Mizuhopecten yessoensis

Some authors claim RNAseq is not necessary to find good reference genes:

RNA-Seq is not required to determine stable reference genes for qPCR normalization

But, as you already have the RNAseq, it doesn't hurt, either.

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written by h.mon h.mon

Thank you so much for these references! they're exactly what I needed.

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written by marina.wakid • marina.wakid

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