Reverse transcription and amplification experiments with low abundance nucleic acids

Summary

This method uses only one recombinant enzyme, rTth DNA polymerase, which allows for simultaneous reverse transcription and amplification in a single reaction, thus eliminating the need for two different buffer systems.

Operation method

one-step method

Materials and Instruments

Nucleic Acids
dNTP MnCl2 MgCl2 BSA rTth DNA polymerase
Water bath Incubator

Move

1. Pretreat slides with peroxidase, proteinase K and DNAase.

2. Configure the one-step reaction system as follows (total volume 100 μl)
(1) 0.5 μl 100 μmol/l forward primer (final concentration 0.5 μmol/l)

(2) 0.5 μl 100 μmol/l reverse primer (final concentration 0.5 μmol/l)

(3) 6 μl 3 mmol/l 4dNTP mixture (final concentration 0.12 mmol/l)

(4) 2 μl 10 mmol/l _MnCl2 (final concentration 0.2 mmol/l)

(5) 10 μl 25 mmol/l MgCl₂ (final concentration 2.5 mmol/l)

(6) 2 μl 10×rTth transcription buffer (final concentration 0.2×)

(7) 8 μl 10× chelation buffer (final concentration 0.8×)

(8) 10 μl 1.7 mg/ml BSA (final concentration 0.17 mg/ml)

(9) 2 μl 2.5 U/ml rTth DNA polymerase (final concentration 9.05 U/ml)

(10) 29 μl DEPC-treated water
3. Add 8 μl (if using 3-well slides) or 12-20 μl (if using single-well slides) of in situ PCR amplification system to each well with a 20 μl pipette tip, which covers the entire surface of the well.

4. Place a 20 mm × 60 mm coverslip on each slide and carefully seal the edges of the coverslip with nail polish.5. On a 92°C heating block, warm the slide for 90 s. Then transfer to a thermal cycler.

6. Replace the in situ PCR reaction system with the one-step reaction system prepared in step 2.
7. Perform reverse transcription according to the following temperature cycling program:

(1) 1 cycle: 15 min, 70°C

(2) 3 min, 92°C

(3) 15 min, 70°C

(4) 3 min, 92℃

(5) 15 min, 70℃

8. Perform in situ PCR according to the following temperature cycling program:

(1) 29 cycles: 1 min, 93°C (denaturation)

(2) 1 min, 53°C (annealing)

(3) 1 min, 72°C (extension)

(4) Final step: indeterminate, 4°C (maintenance)

9. Remove the slide from the thermal cycler, immerse it in 100% ethanol for ≥5 min to dissolve the nail polish, and use a razor or other knife to skid the coverslip and scrape off the nail polish residue so that a new coverslip can be placed flat during the hybridization/detection step.

10. incubate slides on a 92°C heating block for 1 min, then soak in 2×SSC for 5 min at room temperature. slides can be stored at 4°C for 2-3 weeks.

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