Ribonuclease protection (mapping of RNA with ribonuclease and radiolabeled RNA probes)
Ribonuclease protection (mapping of RNA with ribonuclease and radiolabeled RNA probes)
Ribonuclease protection analysis is used to measure the abundance of specific mRNAs and to map their topological heterogeneity. This method involves hybridization of the test RNA to a complementary radiolabeled RNA probe (riboprobe), followed by cleavage of the unhybridized sequence by one or more single-strand-specific ribonucleases. This experiment is derived from "Molecular Cloning Laboratory Guide, Third Edition", translated by Huang Peitang et al.
Operation method
Ribonuclease protection (mapping of RNA with ribonuclease and radiolabeled RNA probes)
Principle
Ribonuclease protection analysis is used to measure the abundance of specific mRNAs and to map their topological heterogeneity. This method involves hybridization of the test RNA to a complementary radiolabeled RNA probe (riboprobe), followed by cleavage of the unhybridized sequence by one or more single-strand-specific ribonucleases.
Materials and Instruments
RNA polymerase DNase I Protease K RNA enzyme inhibitor RNA enzyme digestion mix Carrier RNA Ribonucleotide Standard RNA RNA RNA to be tested UTP RNA probe Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate DTT hybridization buffer Phenol Chloroform RNA gel spiking buffer SDS Sodium acetate TE Transcription buffer Trichloroacetic acid Polymerase dilution buffer
Denatured polyacrylamide gel Water bath Whatman 3 MM filter paper or equivalent
1. Buffers and solutions
Ammonium acetate (10 mol/L)
DTT ( 0.2 mol/L)
Hybridization buffer (for RNA) (40 mmol/L PIPES (pH 6.8), 1 mmol/L EDTA (pH 8.0), 0.4 mol/L NaCl, 80% deionized formamide, using the disodium salt of PIPES, pH adjusted with 1 mol/L hydrochloric acid).
Phenol: chloroform (1:1, V/V)
RNA gel spiking buffer (95% deionized formamide, 0.025% (m/V) bromophenol blue, 0.025% (m/V) xylene cyanide blue FF, 5 mmol/L EDTA (pH 8.0), 0.025% (m/V) SDS)
SDS ( 10% m/V)
Sodium acetate (3 mol/L, pH 5.2)
TE ( pH 7.6)
10X transcription buffer (0.4 mol/L Tris-Cl (pH 7.5), 0.1 mol/L NaCl, 60 mmol/L MgCI2, 20 mmol/L spermidine, stored at -20°C in portions)
Trichloroacetic acid (1% and 10% TCA)
2. Enzyme and buffer
Phage-encoded DNA-dependent RNA polymerase
DNase I (1 mg/ml, RNase-free tryptic DNase)
Polymerase Dilution Buffer
Protease K (10 mg/ml)
RNAase inhibitor, on ice
RNAase digestion mixture (300 mmol/L NaCl, 10 mmol/L Tris-Cl (pH 7.4), 5 mmol/L EDTA (pH 7.5), 40 μg/ml RNase A, 2 μg/ml RNase T1 in 10 mmol/L Tris-Cl (pH 7.5), 15 mmol/L NaCl). 10 mg/ml RNase A (calf pancreas RNaae), 1 mg/ml RNase T1 with 10 mmol/L Tris-Cl ( pH 7.5), 15 mmol/L NaCl, gel)
Denatured polyacrylamide gel (containing 8 mol/L urea)
3. Nucleic acids and oligonucleotides
Vector RNA (1 mg/ml)
Plasmid DNA or linear target DNA for template preparation
Ribonucleotide
Standard RNA
RNA to be tested
UTP (100 μmol/L)
4. probe
RNA probe
5. Radioactive components
[ α-32P ] UTP (10 mCi/ml, 800 Ci/mmol)
6. Specialized equipment
Water baths preset to 30°C, 85°C, 95°C and appropriate recharacterization temperatures
Whatman 3 MM filter paper or equivalent
II. Methods
Preparation of randomly labeled single stranded RNA probes
1. Preparation of linear DNA templates
(1) Preparation of template from plasmid DNA
① Linearize 5~20 μg of plasmid by cutting within or downstream of the cloned DNA sequence with a 5-fold excess of an appropriate restriction endonuclease. The distance between the linearized ends and the promoter should be 200~400 bp. Make sure that the restriction enzyme used does not directly separate the promoter from the target sequence. Since the phage-encoded RNA polymerase requires a 3' end to initiate transcription, choose a restriction enzyme that produces a flat end or a 5' protruding end.
② After the digestion reaction, take a small amount of the reaction solution and analyze it by agarose gel electrophoresis. Make sure that no trace of cyclic plasmid is visible, otherwise, add appropriate amount of enzyme and continue digestion until no cyclic plasmid is detected.
③ Phenol: Chloroform was extracted twice to purify the linear DNA, which was then precipitated using ethanol. The precipitate was washed with 70% ethanol and solubilized in TE solution pH 7.6 at a concentration of 1 μg/μl.
(2) Preparation of template for target DNA amplification
① Amplify double-stranded DNA templates of 100~400 bp in length by PCR (Schowalter and Sommer 1989; Bales et al. 1993; Davis et al. 1997).
The PCR amplification products were detected by agarose or polyacrylamide gel electrophoresis to ensure that the fragment sizes were correct.
(iii) Phenol: purified linear DNA was extracted twice with chloroform and then precipitated with ethanol. The precipitate is washed with 70% ethanol and dissolved in TE solution pH 7.6 at a concentration of 1 μg/μl.
2. Mix the following solutions in sequence and preheat to room temperature unless otherwise indicated.
0.5 μg linear DNA template
1 μl 0.2 mol/L DTT
2 μl nucleic acid solution
1 μl 100 μmol/L UTP
50~100 μCi [ α-32P ] UTP (10 mCi/ml, 800 Ci/mmol)
Replenish with water to 16 μl
2 μl 10X transcription buffer
24 units RNAase inhibitor (on ice)
15-20 units phage RNA polymerase (on ice)
The sequential addition of the above reagents at room temperature prevents precipitation of DNA by spermidine and Mg2+ in the transcription buffer, and prevents inactivation of the RNAase inhibitor by the high concentration of DTT.
Hold the mixture at 37℃ for 60 min.
3. At the end of the above reaction, add about 1 μg of 1 unit of RNAase-free DNA enzyme and continue holding at 37℃ for 10 min.
4. Dilute the reaction solution with TE buffer (pH 7.6) to 100 μl, and take 1 μl to measure the total radioactivity and the radioactivity precipitated by TCA. Calculate the weight and specific activity of the RNA probe synthesized by the reaction from the proportion of radioactivity of the TCA-precipitable material incorporated into the reaction.
5. After removing 1 μl of the solution from step 4, add 10 μl of 1 mg/ml carrier RNA to the remaining diluted reaction solution. phenol: Chloroform extracts the reaction solution, the aqueous phase is transferred to a new tube, and 10 μl of 10 mol/L ammonium acetate 300 μl of ethanol is added to precipitate the RNA. store the reaction solution at -20°C until step 4 is completed.
6. High-speed centrifugation at 4℃ for 10 min was used to recover the RNA. 75% ethanol was used for washing and centrifugation again. Discard the supernatant and dry the RNA at room temperature until no trace ethanol is visible. If the probe is to be further purified by gel electrophoresis (step 7), dilute the precipitated RNA with 20 μl of gel spiking buffer; otherwise, dilute the precipitated RNA with 20 μl of TE buffer (pH 7.6).
7. Purify the probes by electrophoresis on a pre-prepared polyacrylamide gel containing 8 mol/L urea. Highly specific active probes should be used in the near future to avoid radiochemical damage to the RNA.
8. Combine each RNA to be tested with a standard RNA nucleic acid probe ( 2X105-10X105 cpm, 0.1-0.5 ng). Add 0.1x volume of 3 mol/L sodium acetate (pH 5.2) and 2.5x volume of ice ethanol. The mixture was stored at -20°C for 10 min and centrifuged at 4°C for 10 min to recover the RNA. 75% ethanol was used to wash the precipitate. The precipitate was washed with 75% ethanol. The ethanol was carefully discarded and the precipitate was dried at room temperature until the ethanol was completely evaporated.
9. Dissolve the RNA in 30 μl of hybridization buffer, blowing the solution up and down several times to ensure complete dissolution of the precipitate. 
10. Hold the hybridization mixture at 85°C for 10 min to denature the RNA, then quickly transfer it to the back of a thermostat set at the recovery temperature or to a water bath and hold for 8 to 12 hours.
11. Cool the hybridization mixture to room temperature, add 300 μl of RNAase digestion mix, and digest for 60 min at 30℃.
12. Add 20 μl 10% SDS and 10 μl 10 mg/ml proteinase K to terminate the reaction. Add 20 μl of 10% SDS and 10 μl of 10 mg/ml Proteinase K. Hold the reaction mixture at 37℃ for 30 min.
13. Add 400 μl phenol: chloroform, shake vigorously for 30 s, centrifuge for 5 min at room temperature to separate the phases.
14. Transfer the upper phase to a new tube, being careful to avoid contact with the interface between the two phases.
15. Add 20 μg of carrier RNA and 750 μl of cold ethanol. Mix the solutions and place at -20°C for 30 min.
16. Recover the RNA by high speed centrifugation at 4°C for 15 min. Carefully discard the ethanol and wash the precipitate with 500 μl of 170% ethanol. Centrifuge again.
17. Carefully discard ethanol and dry at room temperature until trace ethanol evaporates.
Gel electrophoresis analysis of anti-RNAase hybrids
18. Resuspend the precipitate with 10 μl of gel spiking buffer.
19. Heat the nucleic acids at 95°C for 5 min and quickly place on ice. Centrifuge briefly to collect the sample at the bottom of the tube.
20. Analyze labeled nucleic acids by electrophoresis using a thin polyacrylamide gel containing 8 moI/L urea.
21. When the tracer dye has migrated the appropriate distance in the gel, turn off the power and remove the electrophoresis unit. Slowly remove the large glass plate from the gel by gently prying off one corner of the plate. Cut off the corner of the gel to identify the direction.
22. Transfer the glass plate with the gel to a tray containing an excess of 10% TCA. Gently shake or rotate the tray at room temperature for 10 min.
23. Pour off the 10% TCA solution and replace it with an excess of 1% TCA. gently shake or rotate the tray at room temperature for 5 min.
24. Pour off the 1% TCA solution and rinse the fixed gel with distilled deionized water. Remove the glass plate with the gel from the tray and place it on a flat surface. Remove excess water from the edge of the gel with a piece of paper.
25. Cut a piece of 30 mm filter paper 1 cm larger than the edge of the gel, place it over the gel, and invert the glass plate.
26. Remove the glass plate and place the gel in a gel dryer at 60°C for 1 to 1.5 hours.
27. Establish the autoradiographic image of the gel. By densitometric or phosphor photographic scanning, it is also possible to cut off the part of the gel containing the corresponding fragment and count it using a liquid flash meter.