Nuclease protection assay
Nuclease protection assay
Ribonuclease protection assay (RPA) is a new method for quantitative analysis of mRNA developed in the last decade. The basic principle is to hybridize the labeled specific RNA probe (32P or biotin) with the RNA sample to be tested, and the labeled specific RNA probe specifically binds to the target gene according to the principle of base complementarity to form double-stranded RNA; the unbound single-stranded RNA will be digested by RNAase A or RNAase T1 to form oligonucleic acid, and the target gene to be tested will form double-stranded RNA by combining with specific RNA probe, so the method is free from digestion by RNAase. Double-stranded RNA is protected from digestion by RNAase, so the method is named RNAase protection assay. For 32P-labeled probes, the hybridized duplexes were subjected to denaturing polyacrylamide gel electrophoresis, and the signals of the protected probes were detected by radioautoradiography or phosphor-screen imaging system; for biotin-labeled probes, the hybridized duplexes were electrotransferred to nylon membranes after denaturing polyacrylamide gel electrophoresis, and the signals were detected using the Streptavidin- Horseradish Peroxidase (HRP) and chemiluminescence substrate. HRP) and chemiluminescent substrates were used to bind to the biotin-labeled probes on the membrane, and the hybridization signals were detected by X-ray film or chemiluminescent image analyzer.
Operation method
Ribonuclease protection
Materials and Instruments
Nucleases Move 1. Create 20 μl of reaction complex in a microcentrifuge tube under high pressure as follows: For more product details, please visit Aladdin Scientific website.
ATP CTP GTP MUTP EDTA NaCl Formamide Tris-HCl RNase A RNase T1 RNasin DTT UTP T7RNA polymerase DNaseⅠ Saturated Phenol Chloroform Yeast tRNA NaAc Anhydrous Ethanol SDS Protease K Isopropyl Alcohol Acrylamide Methylenebisacrylamide TBE Urea Urea Ammonium Peroxynitrite TEMED
Low Temperature Centrifuge Constant Temperature Incubator Constant Temperature Water Bath Electrophoresis Instrument Cling Film
(1) 4 μl 5× transcription buffer
(2) 1 μl 200 mmol/ 3NTP mixture
(3) 10 μl ［ α-32P］CTP
(4) 1 μl placental nuclease inhibitor
(5) 1 μl 1 mg/ml template DNA
(6) 1 μl phage RNA polymerase
(7) The SP6 RNA polymerase reaction is held at 40°C for 30-60 min. for T7 or T3 RNA polymerase, incubate at 37°C.2. Add 5 μg or 10 U of Enzyme I without RNAase and incubate at 37°C for 15 min.
3. Add 2 μl of 10 mg/ml tRNA, rehydrate to 50 μl, and extract with phenol/chloroform/isoamylase.
4. To the aqueous phase, add 200 μl of 2.5 mol/l ammonium acetate and 750 μl of anhydrous ethanol, mix well, and then leave on ice for 15 min, and then centrifuge for 15 min at 4°C in a microcentrifuge.5. Re-dissolve in 50 μl of water and re-precipitate twice as described in step 4, the final precipitate was washed with 75% ethanol/25% 0.1 mol/l pH 5.2 sodium acetate buffer, the precipitate was allowed to air-dry and dissolved in 100 μl of hybridization solution, and 1 μl of it was counted.6. RNA was precipitated with ethanol and dissolved in 30 μl of hybridization solution containing 5×105 cpm RNA probe, denatured by heating at 85℃ for 5 min, transferred to the set hybridization temperature (30~60℃), and incubated for more than 8 hours.
7. Add 350 μl of nuclease digestion buffer containing 40 μg/ml nuclease A and 2 μg/ml nuclease T1, and incubate at 30℃ for 30~60 min.
8. Add 10 μl of 20% SDS and 2.5 μl of 20 μg/ml of proteinase K. Incubate at 37℃ for 30~60 min.
9. Extract with 400 μl phenol/chloroform/isoamyl alcohol, transfer the aqueous phase into a centrifuge tube containing 1 μl 10 mg/ml tRNA, add 1 ml ethanol to precipitate, air-dry, and then dissolve in 3~5 μl RNA spiking buffer.
10. Heat denaturation at 85°C for 3 min, electrophoresis in a denatured polyacrylamide/urea gel (serial gel), and analyze the results by radiation autoradiography with a sensitized screen overnight.