RNA isolation for Microarray
Description
RNA extraction using TRI REAGENT. This method gives ample amout of RNA.
Procedure
It is 3 days procedure.
Day 1:
1. Harvest the cells and centrifuge at 2000 rpm for 5-10 minutes and pour off the media.
2. Wash the cell pellet with PBS and add 10 ml TriReagent solution.
3. Incubate at Room Temperature for 5 min and during incubation pass the cell suspension through Pipette.
4. Collect the solutions in fresh 50 ml tubes and incubate at RT for 5 min (to ensure nucleoprotein complex dissociation).
5. Add 1 ml of 1-Bromo-3-chloropropane (BCP)(for per 10 ml TriReagent) and shake vigorously for 15 sec.
6. Incubate at RT for 2-15 minutes (Average 10 minutes).
7. Centrifuge homogenate at 3200 rpm for 30 min to 1 hr at 4oc.
8. After centrifugation there will be two phase:- Lower red Phenol phase and upper colorless RNA aqueous Phase.
9. Transfer upper aqueous phase to fresh tube but leave lower 1 ml of aqueous phase.
10. Add 5 ml of isopropanol (per 10 ml of TriReagent)
11. Shake vigorously and leave at -20oC overnight.
Day 2:
1.Centrifuge at 3,200rpm (12,000g) for 30 min - 1 hr at 4 oC.Take off most of water phase by pipette, leave about 1ml in the tube.
2. Add 10ml of 70% ice-cold ethanol (from the freezer!)/10ml of TriReagent used.
3.Incubate the RNA at -20 oC for 30 minutes.
4.Centrifuge RNA at 3,200rpm (12,000g) for 15 min at 4 oC.
5.Carefully pour off the ethanol, add 1ml 70% ethanol into tubes and gently flick the tube.
6.Try to collect all RNA, might add additional 0.5ml 70% ethanol to wash the tube.
7.Transfer this RNA into eppendorf tube, resuspend carefully and spin it again in microcentrifuge at 4 oC at 11500 rpm for 30 minutes to sediment RNA.
8.Pour off ethanol; tilt the tube downward to allow drain excess of ethanol. Do not dry!
9.Use RNAase free filter paper (soaked in ethanol and dried under the hood). Do not touch the RNA pellet.
10.Add 250ul of HPLC grade water. Dissolve RNA and allow it to stay at RT for 5 min.
11. Add 25ul of 3M Sodium acetate buffer and then 625 ul of 100% ethanol from the freezer.
12.Leave overnight in the freezer at -20oC to precipitate.
Day 3:
1.Spin RNA again in micro centrifuge at 40C (10min) to sediment RNA.
2. Pour off ethanol; tilt the tube downward to allow drain excess of ethanol. Use RNAase free filter paper (soaked in ethanol and dried under the hood). Dry precipitate for 5min.
3.Resuspend the RNA pellet in 250ul of HPLC grade water. Place on ice.
4.Do not shake vigorously of pipette RNA.
5. Determine concentration of RNA content.
6.Read absorption at 260 and 280 in spectrophotometer.
7.Quantitative analysis: Abs 260 x 400 x 40 = amount of RNA in ug/ml
Recipes
TriReagent (MRC)
1-Bromo-3-chloropropane (BCP)(MRC)
3M Sodum acetate buffer (Sigma)
iso propanol
100% ethanol
HPLC grade water
Supplies
Tips
Submitted by: sbafna
