Isolation of total RNA from mouse muscle

1. Excise quadriceps femurs, gastronemicus, and isoleus from mouse leg. Put in lN2 and then put in microfuge tube with hole in top. Drop in lN2

2. Crush in caulderizer which has been coole din lN2

3. Put crushed muscle in Trizol. Use 1ml Trizol per 50mg muscle

4. Homogenize with sonicator or polytron while keeping tubes on ice

5. Centrifuge @ 12,000X g for 10 minutes @ 4 C to remove extracellular material

6. Incubate @ RT 5 minutes

7. Transfer supernatant to new tube. Add 0.2mL chloroform per 1ml Tirzol used. Mix vigorously 15 seconds. Incubate 3 minutes @ RT

8. Centrifuge @ 12.000X g 15 minutes at 4 C

9. Remove aqueous phase and put in clean tubes, Add 0.5 ml isopropanol per 1ml Trizol used initially. Incubate 10 minutes@ RT.

10. Centrifuge @ 12,000xg 10 minutes @ 4C

11. White pellet should be visible. Discard supernatant. Wash pellet with 1ml 75% EtOH per 1ml Trizol

12. Mix by vortexing

13. Centrifuge @ 7500xg 5 minutes @4C

14. Discard supernatant and air dry pellet until it become invisible

15. Resuspend in DEPC-treated water