Difference between revisions of "Purification of miRNA and mRNA with TRIzol"

Latest revision as of 21:35, 29 December 2014

This was suggested by Invitrogen Technical Support, since the PureLink RNA prep kit is likely to lose much of the miRNAs. Ideally the PureLink mRNA or miRNA kit would be used. This protocol is largely taken from the TRIzol instruction manual available here

Materials

• TRIzol
• Chloroform
• RNAse-free glycogen (Life Technologies catalog #10814010, 20 ug/uL)
• Isopropanol
• 75% Ethanol
• Heat block set to 55-60C

Protocol

1. Prepare samples in 1 mL TRIzol either from cells or from tissues lysed in the homogenizer. These lysates can be stored indefinitely at -80.
2. Thaw and site at room temperature for ~5 minutes.
3. Add 200 uL chloroform to the tube, mix vigorously by hand for 15s.
4. Incubate 2-3 minutes at room temperature.
5. Centrifuge 15 min at 12 000g (11 000 RPM) at 4C to separate the phases.
6. Carefully remove the upper aqueous phase by angling the tube at 45 degrees. Avoid drawing up any of the interphase or organic layer.
7. Place into fresh tubes
8. Add 10ug of RNAse-free glycogen (0.5 uL) to each tube
9. Add 0.5 mL of isopropanol to tube and incubate at room temperature for 10 mins
10. Centrifuge 15 min at 12 000g (11 000 RPM) at 4C to precipitate the RNA.
11. Carefully remove the supernatant, making sure not to disturb the (often invisible) pellet.
12. Rinse with 1 mL of 75% ethanol. Pellet can be stored at this stage at -80
13. Vortex briefly to mix, then spin at 7500g (8400 RPM) for 5 minutes at 4C. Aspirate the wash
14. Air dry the pellet for 5-10 minutes.
15. Resuspend the pellet in 50 uL RNAse free water by pipetting up and down several times through a pipet tip.
16. Incubate at 55-60C in a heat block to solubilize.
17. Nanodrop and store at -80. A260/280 ratio should be 1.6-1.8.