Can someone help me by solving this issue?
I have a cell culture of THP1 cells and I need to extract RNA but the cells are frozen and they have been washed by PBS and 0.1 M BSA is added. The RNA is used for reverse transcription and then qPCR is going to be performed.
What is the best way to deal with the frozen cells before we start extracting?
Should the BSA be removed? Or should I just scrape the cells and pipette the mixture out?
It is better to extract RNA just after you pellet the cells by centrifugation, without washing them in PBS. Or to snap freeze the pellets by liquid nitrogen, then store them at -80 and later extract RNA by lysing the pellet in Trizol following manufacturer's protocol.
Frozen cells when you are trying to harvest them for RNA, it is always advisable to take the cells out either from liquid N2 or -80 and thaw them in a water bath (37 degrees) for 4-5 min. 2 steps you can follow:
1. culture them minimum for 1-2 passages by adding the entire thaw mixture to 3 ml of respective media in a 60mm dish. once the cells attain morphology and are confluent enough.
2. If you would like to harvest the cells immediately, thaw the cells and collect them in a 15ml conical flask, centrifuge them. Then you definitely need to wash with PBS (Not BSA in PBS). This washing step is necessary to remove the DMSO in freeing media as well as FBS which is present in culture medium. After the PBS wash, centrifuge them, remove the supernatant and add 1ml of trizol solution and proceed with RNA extraction steps.
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