RNA extraction
1.
2.
3.
4.
Count cells
Pellet approx 10 x 106 cells (genrally 10 mL of medium) by centrifugation.
Lyse cells in 1 mL Trizol per 5-10 x 106 cells by repetitive pipetting.
Transfer to as flat top RNA free tube (samples can be stored like in -70C for
1 month).
5. Incubate at room temp for 5 min
6. Spin at 12000 x g at 4C for 10 min
7. Collect supernatant and transfer to a new tube.
8. Add 0.2 mL chloroform per 1 mL of Trizol used.
9. Shake tubes by hand (mixing vigorously) for 15 seconds.
10. Incubate at room temp for 2-3 min
11. Centrifuge samples at 12,000g for 15 min at 4C.
12. Transfer the colourless upper aqueous phase to a fresh tube (set pipette to 400
µL).
13. Add 0.5 mL of isopropyl alcohol per 1 mL Trizol initially used - mix by
inversion)
14. Incubate at room temperature for 10 min.
15. Centrifuge samples at 12,000g for 10 min at 4C.
16. Remove supernatant and discard it.
17. Wash pellet with at least 1mL 75% ETOH per 1 mL Trizol initially used. Mix
by vortexing.
18. Centrifuge samples at 7500g for 5 min at 4C.
19. Remove supernatant and discard it.
20. Briefly air dry (no longer than 5-10 min) Do not let the pellet dry completely
as this will decrease its solubility.
21. Dissolve RNA in 50 µL Rnase-free water (if it doesn’t dissolve place on
heating block for 10 min at 55-60C).
22. Treat samples with DNA-free kit. First add 5.5 µL of 10x DNase sample
buffer to sample.
23. Add 1 µL of DNase 1, mix by flicking, and give a brief spin in centrifuge.
24. Incubate in water bath at 37C for 30 min.
25. Add 5.5 µL of DNase Inactivation reagent (this slurry needs to be vigorously
vortexed before using).
26. Incubate tube for 2 min at room temperature. Flick tube once in this time to
mix.
27. Centrifuge tube at 10000g for 1 min at 15C to pellet the DNA Inactivation
agent. Transfer supernatant to new tube.
RNA concentration determinations:
Measure absorbance at 260 nm and 280 nm using UV spectrophotometer and
calculate difference.
1. Add 79 µL of sterile water to cuvette and blank at 260 nm. The change
wavelength to 280 nm and read again.
2. Then add 1 µL of RNA - read at 260 nm then 280 nm.
3. Calculate A260/A280. If less than 1.65 this indicates a poor yield of RNA.
4. For calculating concentration of RNA in sample (in µg/mL) = A260 x dilution
factor (80) x 40.
5. Next calculate so that 2 µg of RNA per sample is loaded for making cDNA.
NOTES:
 Always wear gloves
 Always use clean glassware, ideally DEPC-treated.
 Keep RNA on ice as much as possible as RNases are activated at room
temperature.