cell culture RNA extraction - cell culture RNA extraction (Feb/18/2004 )
If anyone have a protocol for total RNA isolation (extraction) from cell cultures, please let me know! You may send it to me at this address: firstname.lastname@example.org or here in the forum.
There are tons of protocols for total cellular RNA extraction. check here
You can also use RNA extraction kits or reagents such as TriReagent or RNAzol which are very simple to use.
We use TRIzol to extract RNA from cells and it is very successful.
Just spin your cells down, add 1ml of TRIzol and resuspend the cells in this. Incubate at room temp for 5-10 min then add 0.2ml of chloroform to the solution. Again incubate at room temp for 2-3 min then centrifuge at 4 degrees celcius at 12,000 rpm for 15 mins.
Take your supernatant and discard the pellet. To the supernatant add approx. 3 volumes of Isopropanol (i.e. if you have 0.2 ml of supernatant, add 0.6ml of isopropanol) and incubate at room temp for 10 min. Centrifuge this again at 4 degrees at 12,000 rpm for 15 min.
This time, discard the supernatant. Wash the pellet in 1ml of 75% EtOH and mix gently. Centifuge this for 5 min at 7,500 rpm at 4 degrees then remove supernatant and allow pellet to dry for 5-10 min.
Finally resuspend your RNA pellet in 40-60 microlitres of RNAse free dH2O. Store at -70 degrees until ready to use.
I've used severla methods for total RNA isolation and at least i find ta the trizol method is avery good one and not very expensive. it is from invitrogen.
I think TRizol works fine with large number of cells. But what if the cell number is not big enough such as 0.1 million or much less. Do anyone have good idea how to deal with isolating total RNA from small number of cells?