Primer extension experiment
Primer extension experiment
Primer extension analysis was used to determine the location and quantify the amount of the 5'-end of a specific RNA. To end the labeled oligonucleotide hybridization, the RNA and the deoxyribonucleotides present in the reverse transcription were utilized as primers. The RNA is therefore reverse transcribed into cDNA, which is analyzed by denaturing polyacrylamide gel electrophoresis. cDNA length reflects the number of nucleotides between the base of the labeled primer and the 5'-end of the RNA, and the amount of gene product is proportional to the amount of targeted RNA.
Operation method
Primer extension experiment
Materials and Instruments
RNA Move 1. Add the following reagents sequentially to create a reaction mixture (final volume 10 μl) for labeling the primers. For more product details, please visit Aladdin Scientific website.
T4 Polynucleotide Kinase dATP Sodium Acetate Ethanol EDTA RNaseA Phenol Chloroform
Constant temperature incubator NAP-5 column -70°C refrigerator Low temperature centrifuge
(1) 2.5 μl H2O
(2) 1 μl 10×T4 polynucleotide kinase buffer
(3) 1 μl 0.1 mol/l DTT
(4) 1 μl 1 mol/l spermidine
(5) 1 μl of 50~100 ng/μl hooked nucleotide primer
(6) 3 μl 10 μCi/μl [ γ-32P ]ATP
(7) 0.5 μl 20~30 U/μl T4 polynucleotide kinase
(8) incubate at 37℃ for 1 h.
2. Add 2 μl 0.5 mol/l EDTA and 50 μl TE buffer to terminate the reaction and incubate at 65°C for 5 min.
3. Siliconized 1 000 μl pipette tip was plugged with glass wool to form a small ion exchange crutch, 20 μl AG 50W-X8 resin and 100 μl DE-52 resin were added, and the column was washed with 1 ml TEN buffer.
4. Add the labeled reactants from step 2 to the column, collect the effluent and repeat the column.5. Wash the column with 1 ml, TEN100 buffer, followed by 0.5 ml TEN300 and discard the eluate.6. Elute the labeled oligonucleotide primers with 0.4 ml of TEN600 buffer, collect the effluent, and store in a suitably shielded container at -20°C for later use.
7. Take the RNA sample and mix the following in a microcentrifuge tube (final volume 15 μl):
(1) 10 μl total cellular RNA
(2) 1.5 μl 10× hybridization solution
(3) 3.5 μl radiolabeled oligonucleotides
(4) Ensure that the microcentrifuge tube is sealed and submerge it in 65 °C waterloo for 90 min, remove and cool slowly in air temperature.
8. Add the following extension reaction mix (final volume 30.33 μl per RNA sample) to the microcentrifuge tube in the ice bath:
(1) 0.9 μl 1 mol/l Tris-Cl buffer, pH 8.3
(2) 0.9 μl 0.5 mol/l MgCl2
(3) 0.25 μl 1 mol/l DTT
(4) 6.75 μl 1 mg/ml Actinomycin D
(5) 1.33 μl 5 mmol/l 4dNTP mixture
(6) 20 μl H2O
(7) 0.2 μl 25 U/μl AMV reverse transcriptase9. In a microcentrifuge tube containing RNA and oligonucleotides, add 30 μl of the above extension reaction mixture and incubate at 42°C for 1 h.10. Add 105 μl of RNAase reaction mixture to each microcentrifuge tube and incubate at 37 °C for 15 min.
11. Add 15 μl of 3 mol/l sodium acetate and 150 μl of phenol/chloroform/isoamyl alcohol extraction, transfer the upper aqueous phase into a clean tube, and then add 300 μl of ethanol to precipitate the DNA, the precipitate was washed with 100 μl of 70% ethanol, and pipetted to remove trace ethanol residue, and the precipitate was allowed to air-dry for 5~10 min.
12. The precipitate was redissolved with 5 μl of termination/spiking dye solution, heated at 65°C for 5 min in a water bath, and electrophoresed in a 9% acrylamide/7 mol/l urea gel until bromophenol blue reached the bottom of the gel.
13. The gel was dried and autoradiographed with a sensitized screen.