I am doing an experiment where I have to treat the organoids with insulin, harvest RNA and then perform RNA-seq. But I am not sure how long should the insulin treatment be to see an effect on transcription? Any insights on this would be helpful. Thanks!
Hi Shilpa,
I can not answer your question directly but I can tell you what I did in my experiment few years ago when I was in a Stanford Univ Laboratory.
*we had some epidemiology data that children born to diabetic mothers can have chance of some heart defects in human. I planned experiment, I treated mouse embryonic 12.5 hearts with 4 times higher level of insulin and glucose (than the level seen in amniotic fluid, determined before by Eliza and glucometer respectively). I cultured whole e12.5 hearts in high glucose and high insulin levels for 24h and 48h. And at 12h, I changed the media and replaced by fresh media. *After I isolated single cells by enzymatic treatments and washed the cells with the same media. * I filtered cells thru 70um filters and took these cells to 10X genomics in the same media * I did not sort by flow because I did not want to deprive cells at any stage of the media otherwise gene expression can change. *I got good results.
So I will suggest you to treat cells or tissues for 24h (if not 48h) at least and change medium at 12h. I am sure you will get reliable results, Best wishes
This depends on what you are looking for. At best, you could do a time course to look for early and late responsive genes. Are you interested in direct targets of the insulin signaling pathway or genes responding to changes in glucose or metabolism? For direct transcriptional targets you should consider using cyclohexamide to inhibit translation and thus any RNA increases are direct.
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