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RNase protection

1. Label RNA probe by in vitro translation.

2. Add 1 ml RNAsin inhibitor and 1 ml RNase-free DNase. Incubate for 15 minutes at 37 C.

3. Extract with phenol:chloroform. Add 10 m tRNA as carrier, and precipitate with ethanol. Wash and dry the pellet, resuspend in 100 ml hybridization buffer ( 80% (v/v) formamide in 40 mM PIPES, 0.4 M NaCl, 1 mM EDTA, pH 6.7).

3a. Altenatively, column purify the probe.

4. Precipitate 5-20 mg of cold RNA sample, and resuspend in 24 ml hybridization buffer ( 80% (v/v) formamide in 40 mM PIPES, 0.4 M NaCl, 1 mM EDTA, pH 6.7).

5. Add 1 ml labeled RNA (105 cpm).

6. Hybridize by heating to 95 C for 5-10 minutes, then 55 C for 1-4 hours.

7. Add 350 ml RNAse digestion mix (10 mM Tris pH 7.5, 5 mM EDTA, 300 mM NaCl, 40 mg/ml RNase A, 2 mg/ml RNase T1).

8. Incubate at 37 C for 10-15 minutes.

9. Stop the reaction by adding 10 ml of 20% SDS, 10 ml Proteinase K and incubate at 37 C for 10-15 minutes.

10. Extract with phenol/chloroform. Precipitate with 1 ml ethanol and 1 mg tRNA.

11. Resuspend pellet in 10 ml of formamide loading buffer, boil the sample and run on 8% sequencing gel.