Part I: Design of competitor; Part II: Synthesis, purification and quantification of competitor RNA. This experiment is from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Competitive RT-PCR: Competitor RNA construction assay
Materials and Instruments
Ethanol Double-distilled water DNA template [a-32P]ATP Move I. Materials For more product details, please visit Aladdin Scientific website.
RT-PCR Competitor Construction Kit
1. Buffers, solutions and reagents
Ethanol
Double-distilled water for RNAase removal
2. Nucleic acids and oligonucleotides
DNA template (0.5-1.0ug linearized plasmid template or 0.1-0.5ug PCR template)
3. radioactive complexes
[a-32P]ATP (10mCi/ml) (lCi=37 GBq)
4. Experimental equipment
RT-PCR Competitor Construction Kit (Ambion; includes T7RNA polymerase, 10-fold transcription buffer, 5-fold NTP solution, MnCl2, DNAzyme I, gel priming buffer, probe elution buffer)
II. Methods
1. Prepare the transcription reaction at room temperature by adding reagents in the following order.
10x transcription buffer 2ul
5x NTP solution 4ul
DNA template 1~5ul
[a-32P]ATP (10MCi/ml) 0.4ul
T7RNA polymerase 2ul
2. Flick the tube to mix the reagents, centrifuge briefly and collect the reaction mixture at the bottom of the tube.
3. Incubate the transcription reaction at 37°C for 2 to 4 h. Incubate for 2 to 4 h.
4. Remove 1ul of each sample for later determination of competitor concentration.
5. Add 40ul of probe elution buffer and 200ul of ethanol to the transcription reaction mixture.
6. Incubate at -80°C for 20 min.
7. Centrifuge at maximum speed at 4°C to precipitate the transcription reaction products.
8. Pipette up the supernatant, wash the precipitate with 70% ethanol and dry the precipitate in air.
9. Dissolve the precipitate in 16ul of double-distilled water with RNAase removed.
10. Heat denature the resuspended precipitate (containing the DNA template and transcribed RNA) at 95°C for 2 min.
11. Add 2ul of 10x transcription buffer and 2ul of DNAase I (5 U/ul) with RNAase removed.
12. Flick the tube to mix the reaction reagents, centrifuge, and collect the reaction mixture at the bottom of the tube.
13. Incubate at 37°C for lh.
Competitor purification
14. Add 22ul of Gel Sampling Buffer II to the DNAzyme I reaction.
15. Denature the sample at 95°C for 3 min.
16. Run the sample on a denaturing polyacrylamide (6% acrylamide/8 mol/L urea) gel until bromophenol blue reaches the bottom of the clot.
17. Remove a glass plate from the gel, cover the gel with plastic film, and place on X-ray film for 30 min to 2 h for autoradiography.
18. Develop the X-rays and place them in alignment underneath the gel limb, and cut the full-length RNA transcript from the gel with a scalpel or razor blade.
19. Transfer the clot fragments to a small centrifuge tube. Add 300ul of Probe Elution Buffer and submerge the clumps by simple centrifugation.
20. Incubate overnight at 37°C.
21. Discard the acrylamide gel fragments in the tube and add 600ul of ethanol probe elution buffer containing the RNA transcript.
22. Incubate at -80°C for 20 min.
23. Centrifuge at maximum speed at 4°C for 15-20 min.
24. Wash the precipitate with 70% ethanol and dry in air.
25. Dissolve the precipitate with 15ul of double-distilled water with RNAase removed.
Quantification of competitor RNA
26 Transfer 1ul of transcription reaction and 1ul of purified competitor RNA to a scintillation tube, add scintillation solution and count the number of scintillations.
27. Using the results of the count, calculate the concentration of competitor (umol/ul or copies/ul) using the formula below.
Competitor RNA (umol/L) = (cpm of 1ul of purified competitor RNA ÷ cpm of 1ul of transcription reactant) X (total amount of 500umol/LATP/A in competitor RNA)
Competitor RNA (copies/ul) = (cpm of 1ul purified competitor RNA ÷ cpm of 1ul transcription reactant) X (total amount of 3X1014ATP molecules/A in 1ul competitor RNA)
28. Competitor should be stored at a concentration of not less than 6X1010 copies/ul or 0.1umol/L at -20°C for up to 1 year.