cell culture RNA extraction
Posted 18 February 2004 - 12:00 AM
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.
Posted 16 March 2004 - 02:43 AM
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.
Posted 16 April 2004 - 01:43 AM
Posted 16 April 2004 - 05:11 AM
Edited by alex_osu3, 16 April 2004 - 05:11 AM.