In vitro transcriptional synthesis of single-stranded RNA probes
Not only is it easier to prepare specific single-stranded RNA probes than DNA probes, but they also generally produce stronger signals in hybridization reactions than DNA probes with the same specific activity, presumably due to the inherent higher stability of the heterodimeric strand containing RNA (Casey and Davidson 1977). Although DNA probes are still commonly used for Northern and Southern hybridization, radiolabeled RNA is currently the probe of choice. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
In vitro transcription synthesis of single-stranded RNA probes
Principle
Not only is it easier to prepare specific single-stranded RNA probes than DNA probes, but they also generally produce stronger signals in hybridization reactions than DNA probes with the same specific activity, presumably due to the inherent higher stability of the heterodimeric strand containing RNA (Casey and Davidson 1977). Although DNA probes are still commonly used for Northern and Southern hybridization, radiolabeled RNA is now the probe of choice when analyzing transcription of mammalian genes. Because of its durability and ease of control, RNAase A can be used to digest RNA-RNA heterodimers without the need for a specific S1 nuclease to digest DNA-RNA hybrid molecules, and it can be used in a wide range of concentrations without affecting the results of the experiment (Zirmetal. 1983; Mehonetal. 1984).
Materials and Instruments
Restriction endonuclease RNA polymerase Pancreatic DNAase I Template DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate Bovine serum albumin DTT Ethanol Phenol Chloroform Placenta RNAase inhibitor Sodium acetate Transcription buffer rNTP solution
Agarose gel Microtubes Sephadex G-50 columns Water baths
1. Buffers and solutions
Ammonium acetate (10 mol/L)
Bovine serum albumin (2 mg/ml, fraction V, Sigma)
DTT ( 1 mol/L)
Ethanol
Phenol: chloroform (1:1, V/V)
Placental RNAase inhibitor (20 units/μl)
Sodium acetate (3 mol/L, pH 5.2)
10X transcription buffer (400 mmol/L Tris-Cl ( pH 7.5 at 37°C), 60 mmol/L MgCl2, 20 mmol/L spermidine HCl ), 50 mmol/L NaCl)
2. Enzyme and buffer
Appropriate restriction endonucleases
T3, T7, or SP6 phage-dependent DNA RNA polymerase 
Tryptic DNAase I without RNAase (1 mg/ml)
3. gels
Agarose gel (0.8%~1.0%)
4. Nucleic acids and oligonucleotides
rNTP solution containing 5 mmol/L each of rATP, rCTP, and rUTP.
rGTP (0.5 mmol/L)
Template DNA 
5. Radiocomplexes
[ α-32P ] dNTP ( 10 mCi/ml, specific activity 400-3000 Ci/mmol)
6. Specialized equipment
Microcentrifuge tube (0.5 ml)
Sephadex G-50 column, equilibrated with 10 mmol/L Tris-Cl (pH 7.5)
Preheated to 40°C in a water bath
ii. Methods
1. 5 pmol of linear template DNA was prepared by digesting the superhelical plasmid DNA with an appropriate restriction enzyme. a small portion of the digested DNA (100 ng) was analyzed by agarose gel electrophoresis. If necessary, add additional restriction enzymes and continue incubation until no more traces of undigested DNA remain.
2. If it is necessary to use a restriction enzyme such as PstI or SstI that produces a 3' prominence, the digested DNA fragments should be treated with phage T4 DNA polymerase in the presence of four types of dNTP to remove the 3' prominence produced.
3. Purify the template DNA by phenol/chloroform extraction and precipitation with standard ethanol. dissolve the DNA in water to a final concentration of 100 nmol/L (e.g., 200 μg/ml for 3kb plasmid).
4. Bring the first six components listed below to room temperature and mix them in the following order in a sterilized 0.5 ml microcentrifuge tube at room temperature:
Template DNA 0.2 pmol (400 ng for 3 kb plasmid)
Water without RNAase Add to 6 μl
5 mmol/L rNTP concentrate 2 μl
100 mmol/L DTT 2 μl
10X Transcription Buffer 2 μl
2 mg/ml bovine serum albumin 1 μl
10 mCi/ml [ α-32P ] rGTP 5 μl
( Specific activity 400~3000 Ci/mmol)
Flick the outer wall of the tube to mix the components. Then add:
Placental RNAase inhibitor (10 units) 1 μl
Phage DNA-dependent RNA polymerase (approx. 10 units) 1 μl
Tap the outside of the tube wall to mix the reactants, centrifuge for 1-2 s to settle all liquid to the bottom of the tube, and incubate the reactants for 1-2 h at 37°C (T3 and T7 phage-dependent DNA RNA polymerases) or 40°C (SP6 phage-dependent DNA RNA polymerase).
5. (Optional) For full-length transcripts, add 2 μl of 0.5 mmol/L rGTP and incubate for an additional 60 min at a temperature suitable for the polymerase.
6. Add 1 μl of 1 mg/ml Trypsin I without RNAase to terminate the in vitro transcription reaction. Flick the outside of the tube to mix the reagents. incubate the reaction mixture at 37°C for 15 min.
7. Add 100 μl of RNAase-free water and purify the RNA by extraction with phenol/chloroform.
8. Transfer the aqueous phase to a new microcentrifuge tube and separate the radiolabeled RNA from the unwanted small-molecule RNA and rNTP by any of the following three methods:
(1) Purification of RNA by ethanol precipitation
① Add 30 μl of 10 mol/L ammonium acetate to the aqueous phase, mix well, and then add 250 μl of ethanol pre-cooled with ice. After 30 min on ice, collect RNA by centrifugation in a microcentrifuge at 4℃ for 10 min at maximum speed.
② Carefully aspirate as much ethanol as possible, then leave the tube open and place it on the bench for a few minutes to allow the remaining visible ethanol to evaporate. Redissolve the RNA in 100 μl of RNAase-free water.
③ Add 2 times the volume of ice pre-cooled ethanol to the tube and store the DNA at -70°C for later use.
(2) Purification of RNA by centrifugal column chromatography
① Equilibrate a Sephadex G-50 centrifugal column in 10 mmol/L Tris-Cl (pH 7.5) and sterilize it.
② Purify RNA by centrifugal column chromatography.
③ Store the eluted RNA at -70℃ for backup.
(3) Purification of RNA by gel electrophoresis
① Prepare a neutral polyacrylamide gel.
① Prepare a neutral polyacrylamide gel. ② Add appropriate gel sampling buffer to the aqueous phase and purify RNA by gel electrophoresis.
③ Localize RNA by radioactive autoradiography.
④ Purify RNA from gel block by squeezing and soaking.
⑤ Store RNA at -70°C for backup. 
