We have cultured c2c12 cells and will be moving on to the RNA purification portion of the project. Our protocol asks us to transfer cells to a microcentrifuge tube followed by discarding the supernatant. Our issue is that we won't have enough cells to see a pellet as we ran this in a 96 well plate. Can we instead move to the addition of the lysis buffer directly to the 96 well plate and then store until ready to proceed with the RNA extraction at a later date?
Depends on your lysis buffer: if it's "completely destroys RNAse activity", then you're probably ok. If it's "lyses cells to leave lysates compatible with protein analysis", then you're less ok, because as soon as you lyse cells, RNA starts degrading.
I used to use cells for both protein and RNA analyses, and thus would remove media, wash 1x with PBS, add then my RIPA lysis buffer straight onto the cells (on ice), scrape with a cell scraper and then immediately take half the lysate and pipette it directly into trizol. This would be my "RNA half", which I would then freeze on dry ice for later RNA extraction while I got on with preparing a protein half from the remaining lysate.
If you're using something like trizol (phenol + guanidium isothiocyanate) then you can pipette that directly onto washed cells: leave it at RT for ~1-2 mins to dissolve/lyse/denature everything, then pipette mix a few times and transfer to microcentrifuge tubes, then freeze for later RNA isolation.
Alternatively, you can literally freeze the cells themselves: remove media, wash 1x with pbs, remove that, then place the 96 well plate directly on dry ice to snap freeze. Put the lid back on, wrap in parafilm or foil while still frozen (or put in a ziploc bag), store at -80 until needed. Then you can isolate RNA by just adding the lysis buffer (trizol ideally) straight onto the frozen cells and let them defrost while denaturing.
I always try to extract my RNA as the same setting when I lysed my cells. RNAs are very sensitive to degrade so I don't freeze them. But if anyone with more experience can suggest I will try to freeze them to see if this affect my RNA quality or not.
Tahmina Haque you absolutely can freeze RNA: at -80 it's much more stable than at room temp.
Repeated freeze/thaw is not kind to RNA, but mostly only when it's purified and in solution. And you shouldn't need to repeated freeze thaw RNA anyway.
"Isolate, then freeze" is fine, as is "add denaturing lysis, then freeze".
Hi Alfredo,
If there is no obvious apoptosis or morphological change, and RNA extraction is not performed immediately after cell collection, you can discard the culture medium and place the dish upside down in a -80 °C freezer for storage. If there are morphological changes, it's generally better to collect the cell pellet and store it at -80 °C. When ready to proceed, take it out, extract the RNA, and perform reverse transcription immediately to avoid RNA degradation.
Hope this helpful. Feel free to reach out for more details.
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