RNAse Protection Assay

1. DNAse-treat RNA for 1 hr using RQ kit.

2. EtOH ppt RNA

3. Dissolve 10-60 mg RNA pellet in 20 mL hybridization buffer.

4. Add 20,000 - 50,000 cpm of each probe per sample.

5. Heat to 90 ^OC for 10 min.

6. Hybridize overnight at 58 ^OC.

7. The next day, add 300 mL of T1/T2 mixture

50 mL T1/T2 RNAse

950 mL T1/T2 buffer

8. Incubate at 37 ^OC for 1.5-2 hrs.

9. Add 200 mL GTCN / glycogen (1 mL of 1 mg/mL) and 1 mL of cold EtOH.

10. Ppt on dry ice 30 min - 2 hr.

11. Spin max speed 10 min.

12. Throw out supernatant. Dissolve pellet in 12-15 mL stop dye and heat to 90 ^OC for 2 minutes.

13. Run on a 6% denaturing (urea) polyacrylamide gel at 13 watts for 1-2 hrs.

14. Dry gel onto Whatmann paper.

15. Put under film for 1-5 days. Enjoy!