Hi,
I am growing Caco2 cells in 24 Transwell cell inserts. I am planning to induce gut leakage by LPS and want to study gene expressions. My concern is to isolate the RNA from transwell inserts since those are very tiny and hard to use cell scrapper. Any protocol or suggestions would be greatly appreciated
Hi,
I hope the attachment is useful for your reference.
According to the protocol provided in the reference, you can harvest and lysis cells using Buffer RLT directly without any scraping, followed by RNA purification as instructed.
Good luck!
Hi Samhita!
Just to complement Phan Thi Lam Hong 's answer, if you don't desire to perform the RNA extraction the same day, you can trypsinise cells, centrifuge (optionaly resuspend in PBS to wash and centrifuge again) and then freeze the pellets at -80ºC until the day of extraction. Afterwards, in this day you can proceed adding the lysis buffer of RLT buffer (depending on the protocol you usually follow to extract RNA).
Hope it helps!
Best regards :)
Francesc
Trizol (or similar reagents) works nicely for this, add it directly to the trans-well to lise cells and recover RNA. You can then pool multiple wells if desired. Also, RNA is stable in Trizol (different periods depending on the storage temperature), so no need to proceed immediately with the extraction.
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