Analysis of RNA by primer extension.

1. Phosphorylate oligonucleotide probe: a. Oligonucleotide primer (5 – 7 pmoles or 60 ng) 1 μL b. dH2O 6.5 μL c. 10X kinase buffer 1.5 μL d. T4 PNK (~10 U) 1 μL e. [γ-P]ATP (7000 Ci/mmole) 2 μL 2. Stop the kinase reaction by adding 500 μL of TE (pH 7.6) 3. Add 25 μg of carrier RNA. 4. Phenol/Chloroform extract 2X 5. Ethanol precipitate with sodium acetate (pH 5.2) 6. Wash with 70% ethanol and resuspend in 500 μL of TE (pH 7.6) 7. Measure specific activity (should be ~2 x 10 cpm/pmol) 8. Mix 10 – 10 cpm (20 – 40 fmoles) of DNA primer with 0.5 – 150 μg of RNA 9. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol. 10. Precipitate, wash with 70% ethanol and resuspend in 8 μL of TE 11. Add 2.2 μL of 1.25 M KCl and vortex 12. Incubate oligonucleotide/RNA mixture in water bath at appropriate annealing temperature for 15 minutes 13. Supplement primer extension mix with DTT and reverse transcriptase and RNAse inhibitor a. To 300 μL of primer extension mix, add 3 μL of 1 M DTT and reverse transcriptase to a final concetration of 2 U/μL and 0.1U/μL of RNAse inhibitor. Mix gently and store on ice. 14. Remove tubes from waterbath and collect by brief centrifugation 15. Add 24 μL of supplemented primer extension mix to each tube. 16. Mix gently and incubate at 42C for 1 hour 17. Terminate primer extension reaction by adding 200 μL of TE (pH 7.6) 18. Phenol chloroform extract 19. Precipitate with ammonium acetate. Wash with 70% ethanol. 20. Resuspend with 10μL of formamide loading dye 21. Heat at 95C for 8 minutes and immediately place on ice for 1 minute. 22. Run on polyagrylamide gel. Dry. Expose to film or phosphor screen.