1. Total RNA is isolated from individual mouse livers by TRIzol; reagent (Invitrogen) and further purified by RNeasy kit (Qiagen).
2. Add 100 mg of frozen tissue directly to 4 mL of TRIzol and dissociate by homog-enization with a rotating blade tissue homogenizer.
3. Add 2/10 vol of chloroform and shake for 15 s.
4. Let stand for 3 min. Centrifuge at 12,000g for 15 min at 4°C.
5. Take off the supernatant and add it to a polypropylene tube, recording the volume of the supernatant.
6. Add 0.53 vol of ethanol to the supernatant slowly, while vortex mixing. This will produce a final ethanol concentration of 35% (Note 2).
7. Add the supernatant to an RNeasy maxi column, which is seated in a 50-mL centrifuge tube.
8. Centrifuge at 3000g in a clinical centrifuge with a horizontal rotor at room temperature for 5 min.
9. Pour the flow-through back onto the top of the column and centrifuge again. A significant amount of RNA is not captured by the column matrix in the first pass of the RNA containing solution through the column.
10. Discard the flow-through and add 15 mL of RW1 buffer (from Qiagen Rneasy kit) to the column.
11. Centrifuge at 3000g for 5 min.
12. Discard flow-through, then add 10 mL of RPE buffer (from Qiagen Rneasy kit).
13. Centrifuge at 3000g for 5 min.
14. Discard flow-through, and add another 10 mL of RPE buffer.
15. Centrifuge at 3000g for 10 min.
16. Put the column in a fresh 50 mL tube and add 1 mL of DEPC-treated water from the kit to the column.
17. Let stand for 1 min.
18. Centrifuge at 3000g for 5 min.
19. Add another 1 mL of DEPC-treated water to the column.
20. Let stand for another 1 min.
21. Centrifuge at 3000g for 10 min.
22. Aliquot out 400-|L portions of the column eluate into 1.5-mL Eppendorf tubes.
24. Add 1 mL of ethanol to each tube.
25. Let stand for 15 min.
26. Centrifuge at 12,000g at 4°C for 15 min.
27. Wash pellet twice in 75% EtOH, then store at -80°C
28. Resuspend RNA at approx 1 mg/mL in DEPC water.
29. Concentrate to greater than 7 mg/mL by centrifugation on a MicroCon 30 filter unit, centrifuge at 16,000g, checking as necessary to determine the rate of concentration. This step removes many residual small to medium sized molecules that inhibit the reverse transcription reaction in the presence of fluorescently derivatized nucleotides.
30. Determine the concentration of RNA in the concentrated sample. Store at -80°C.
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