RNase Protection Assay

Description
This protocol can be used to quantitate an mRNA of interest if the RNA probe is present in molar excess over the target mRNA. A labeled RNA transcript is hybridized to an mRNA population and any non-hybridized (single stranded) RNAs are digested by RNase A and RNase T1. The hybridized mRNA is then analyzed on an urea/polyacrylamide gel.

Procedure
1. Combine 0.5 ìg poly A+ RNA (or 5-30 ìg total RNA) and 40,000 cpm [32P]UTP-labeled, gel-purified RNA probe (CAUTION! see Hint #1; see Hint #2) in a microcentrifuge tube.

2. Add 0.1 volume of 3 M Sodium Acetate pH 5.2 and precipitate with 2.5 volumes of Ethanol.

3. Incubate for 15 min on Dry Ice.

4. Centrifuge at 4°C for 15 min in a microcentrifuge at full speed.

5. Wash the RNA with 70% Ethanol and centrifuge the sample for 15 min in a microcentrifuge at full speed.

6. Dry the RNA pellet in a speedvac concentrator for about 5 min.

7. Resuspend the RNA in 1 ìl distilled water by incubating it for about 5 min on a shaker at room temperature.

8. Add 10 ìl Formamide Hybridization Buffer to each tube and vortex and centrifuge for 10 sec.

9. Denature 10 min at 85°C.

10. Hybridize for 3 to 4 hr at 45°C (see Hint #3).

11. Add 300 ìl RNase Solution to each tube.

12. Incubate 1 hr at room temperature to digest the unhybridized RNA.

13. Add 20 ìl 10% SDS plus 10 ìl (10 mg/ml) Proteinase K.

14. Incubate 30 min at room temperature.

15. Incubate 30 min at 37°C.

16. Add 350 ìl of Phenol/SEVAG to the sample and centrifuge briefly to separate the phases. Recover the aqueous phase.

17. Add 0.5 ìl (10 mg/ml) carrier tRNA and precipitate with 1 ml (-20°C) 100% Ethanol.

18. Incubate for 15 min on Dry Ice.

19. Centrifuge the sample for 15 min at 4°C in a microcentrifuge at full speed.

20. Wash the pellet with 70% Ethanol and dry in a speedvac concentrator for about 5 min.

21. Resuspend the RNA in 8 ìl Urea loading Buffer.

22. Boil samples for about 1 min and place on ice immediately.

23. Load one-half of the reaction volume (4 ìl) on a 6-10% denaturing acrylamide gel (1X TBE) that has been prerun for about 30 min (see protocol for Running Denaturing Acrylamide Gels).

24. Load denatured, labeled markers (see Hint #4).

25. Run at 33 watts until the Bromophenol Blue band is near the bottom of the gel.

26. Fix and dry the gel before exposing it to film (see protocols for Fixing Gels and Drying Gels).

Recipes
Urea Loading Buffer 0.05% (w/v) Bromophenol Blue
8 M Urea
0.05% (w/v) Xylene Cyanol
1 mM EDTA
20 mM Tris, pH 8.0

RNase Solution 50 Units/ml RNase T1
0.3 M NaCl
You may have to titrate amount of both RNases as high as 8X.
5 mM EDTA
0.05 Units/ml RNase A
10 mM Tris, pH 7.5

Hybridization Buffer (5X) 5 mM EDTA
200 mM PIPES, pH 6.4
2 M NaCl

Formamide Hybridization Buffer 1 mM EDTA
(or 8 ìl Deionized Formamide and 2 ìl 5X Hybridization Buffer)
80% Deionized Formamide (CAUTION! see Hint #1)
40 mM PIPES, pH 6.4
0.4 M NaCl

Phenol/SEVAG 25:24:1 Phenol:Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)

TBE 2 mM EDTA, pH 8.0
89 mM Tris
89 mM Boric Acid

Carrier tRNA (10 mg/ml)

10 mg/ml Proteinase K

10% (w/v) SDS

70% (v/v) Ethanol

3 M Sodium Acetate, pH 5.2

Supplies

Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The RNA probe should be 300 to 900 nucleotides long and not very A-T-rich.

3. This hybridization can go as long as overnight.

4. A pBR322 Msp1-digest can be used.
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RNase Protection Assay