What is the criteria of gene selection from RNA seq data for functional validation (knock out or over-expression)?
I would suggest selecting a cut off of >1.5 fold change (up or down-regulated) and p1. And then consider filtering the list based on relevance of the genes to the cell type/tissue type/disease you are studying. An alternative is to perform pathway analysis of the differentially expressed genes (i.e. those >1.5 fold change, p1) and look at what particular pathways are affected. Are there pathways relevant for your cell/tissue/disease. and then select genes from these pathways for functional validation.
As your question itself stated that you want functional validation, therefore I suggest you look for those unigenes which are suitable according to pre planned work. For instance if I am establishing relationship of secondary metabolite w.r.t altitude. Then I will more focus on secondary metabolites related unigenes. If RNA-seq is showing some secondary metabolites related unigenes are down-regulated at lower altitude, and I am able to show similar result via qRT-PCR, then this analysis will itself establish that plant is under stress condition.
Simon Wyn Jones , Nitesh Kumar Sharma. Thanks for your generous reply. Actually I already have done the RNA-Seq analysis, got DEGs, categorized them into relevant pathways. Also validated RNA seq data by comparing of gene expressions through RT-qPCR. Now i want to pick one or two genes from the RNA-seq results and to verify their effect on cellular physiology and my phenotypics traits by knocking them down (Gene silencing). Now my question it that, should i pick the genes which are common among the pathways of my interest and related the cellular phenotypic traits i am verifying on molecular level?
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