I'm planning to isolate RNA from cells stored in RNAlater with Qiagen mini kit. In one article, I saw we can directly use cells in RNAlater without washing it with PBS (but needs to put a higher volume of lysis buffer). Does anyone has done that?
Hi ! By cells I suppose do you mean in vitro culture ?If you want to use the cultured cells directly , you don't need to put them in RNA later . But if you mean tissues stored in RNA later , you can directly proceed to chop it in pieces and then incubating in lysis buffer (without PBS washes). Hope it helps.
Dear Nirmani, if your cells are stored in RNALater at - 80 C then after thawing suspend them at 2500 g at 4C for 5 minutes then discard supernatant to get rid of RNALater solution and start extraction by adding up the lysis buffer, whereas, if you had stored your cells in the RNALater at -20 C then after thawing suspend them for 10 minutes at 5000 g at 22 C, then discard supernatant and start your RNA extraction. Volume of cells to be stored in RNALater is 1 * 10^5 - 10^6 / ml in 200 ul RNALater, but it is always advisable to get your pellets washed with cold PBS first then freeze them at -80c or -20C by adding up the RNALater. It is the recommended protocol for cell pellets. Hope it helps....
Regards....
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