Dear all,
I need to analyze the expression of 8 specific hypothalamic genes through qRT-PCR technique.
My starting material is represented by mice paraffin embedded brains. Since it is difficult to localize the hypothalamus, I am extracting the RNA from the entire tissue with FFPE RNA Kit (Qiagen).
Do you know a method to enrich my samples with mRNA of my interest?
Thank you.
For RT qPCR, you can use random hexamer/oligo dTs or a mixture of both. I prefer using the BioRad's iScript which has both.
With FFPE tissue, it may be best to use random hexamer as they will be less susceptible to degredation associated with FFPE samples.
The gene-specific enrichment can also work better in certain scenarios but it has its own limitations.
https://www.researchgate.net/post/Oligo-dT_vs_random_Primer_cDNA_synthesis_of_RNA_from_FFPET
https://lifescience.roche.com/en_tr/articles/optimal-primer-design-for-rt-pcr.html
there is a technique called interactome capture that allows you to pulldown mRNAs that interact with a specific RNA of interest...the paper is from Alfredo Castello
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