targeted RNA-Seq best practices

Question

[joneill4x]

What are the best practices for a targeted RNA-Seq experiment?

(GOAL: Test if a specific list genes are differentially expressed in sample1 vs. Sample 2)

How to design the primers that capture the target genes? Whats sort of controls are necessary, if any?

Thank you.

[jkkbuddika]

If you intend to see a selected set of genes are expressed at different levels in two conditions you simply perform qPCR in which case you have to design primers. I usually look at literature to see if there are any published primer sequences for your genes of interest (these are verified primers and should work), if not design by myself.

If this is RNA-seq that you want to do, essentially you prepare libraries (from rRNA-depleted or poly-A selected RNA), sequence, map to a genome of interest, generate count matrices and perform differential gene expression analysis using a tool like DESeq2 or edgeR.

[joneill4x]

Thank you for your reply. I was thinking about something like the following, but I will look into qPCR. Please direct me to any good tutorials if you know of them. Thanks again.

https://www.illumina.com/techniques/sequencing/rna-sequencing/targeted-rna-seq.html

"Targeted Amplicon RNA-Seq" "Provides qualitative and quantitative information for differential expression analysis"

Answer 1

[ivivek_ngs]

Hi, there is another technology that you can use if you don’t want to validate your DE genes panel in another cohort or same but another replicates. This is via multi panels from a technology call NanoString. They can profile few custom panels but also have their own. Probes are already available but also can be custom designed but might be on the expensive end if it’s not RT-qPCR . There is also ddqPCR if I am not mistaken. If NanoString budget is available then check the link below.

NanoString panels for expression profiling.