I will be isolating total RNA from hiPSC cardiomyocytes using a Qiagen kit. Would it be better to freeze the cells straight away, then thaw and lyse, or to add lysis buffer to the cells before freezing?
I would store your cell pellets immediately following harvest by freezing, rather than lyzing before. I recommend that you do a flash freeze by putting your collection tubes in liquid nitrogen before storing samples at -80°C. You can help maintain the integrity of your RNA by adding some RNAlater to your samples. For example, adding 100-200ul. The RNAlater will need to be diluted with PBS so that you cells can pellet down before performing the RNA extraction. When I collect primary cells that will be used for qPCR, I harvest the cells in Eppendorf tubes that are qPCR clean (ie, clean with no RNases or DNases present).
Hi Alisha
- As mentioned by Anne it is always better to freeze cell lysate @ -80C than cell pellets:
- The cell lysis buffer contains GITC which renders your RNA completely stable whereas cell pellets even @ -80C can incur some RNA degradation
- I have stored human cardiomyocytes as lysates @ -80C for many months before extracting RNA without RNA degradation. In contrast the same cells stored as whole cell pellets have demonstrated some RNA degradation in the same time period
Thank you both. The cells are coming directly from plates Anne, sorry I forgot to mention, so I do not have cell pellets. In that case adding lysis buffer before storage may be the best option.
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