Basically, I am comparing gene expression in different neuroinflammatory pathways using mRNA Seq in a transgenic (KO) mouse model. The main goal is too see what genes are compensating during a neuroinflammatroy response.
What other experiments often accompany an RNA seq focused project? Western Blot? IHC? qPCR validation? And what would the purpose of those experiments serve in further elucidating the role of this missing gene and the henceforth compensatory mechanisms?
This is for my Masters thesis project so any advice would be helpful!
Hi,
From rather global approaches you can also go for ATAC seq to check the chromatin remodelling.
After you end up with a couple of candidate genes, you can validate them with RT-PCR. Or you can go directly to the protein level and do WB (if you can purify enough of your cells of interest) or IHC/IF (if you stain the whole tissue or if only a part of cells express your protein).
Later you can define the pathway in-vitro by KD or OE of your protein in WT/KO cells to see the desired phenotype. Finally, you can make the same in-vivo and look for the way to rescue the phenotype by (for example) OE/KO the downstream targets of your candidate gene(s).
Best,
Michail
Ideally both PCR and WB, but if you have budget for only one the latter makes more sense especially if you plan on publishing. For PCR validation, you could randomly choose several genes that are not differentially expressed and some genes that are differentially expressed. Genes of interested could be in the pathways that are pertinent to your investigation i.e. inflammation and the compensatory genes. I will give you an example of how my previous PI represented microarray results to give you an idea. Regarding compensatory genes, it may be interesting to investigate which one of these genes in crucial or whether they are all crucial for the compensation mechanism. You could perhaps make an in vitro model for this i.e. using RNAi or CRISPR but this can be expensive, challenging and take a lot of your time.
Article Unique Transcriptional Profile of Sustained Ligand-Activated...
IHC is great to provide visual context to where proteins are localised and WB is also good for determining protein concentration.
It's worth noting that the relationship between RNA and Protein is not fully understood, and a low mRNA expression doesn't mean the protein is produced in low amounts.
Staining for immune cells with IHC in your cases will allow you to see if there is any apparent changes in neuroinflammation.
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