In situ PCR amplification of DNA and RNA target sequences
In situ PCR amplification of DNA and RNA target sequences
In situ PCR, a technique established by Hasse in 1990, involves performing PCR reactions in tissue cells. It combines the advantages of in situ hybridization with cellular localization capability and highly specific and sensitive PCR technology, and is a promising new technology in the field of cytology research and clinical diagnosis.
Operation method
basic program
Materials and Instruments
Samples Move 1. Place the slide containing the immobilized sample on a heating block at 105°C for 5-120 s.2. Slides were placed in 0.3% H2O2 and incubated overnight at 37°C or room temperature to inactivate endogenous peroxidase activity, then washed once with PBS. For more product details, please visit Aladdin Scientific website.
Hydrogen peroxide PBS Proteinase K RNAase DTT dNTP KCl MgCl2 Tris-Cl
Humidifying cartridges Thermocyclers
3. Take 1 mg/ml of proteinase K diluted in 150 ml of PBS (final concentration 6 μg/ml), immerse the slide in this solution, incubate for 5 min at room temperature, and then observe the cells under a 400× microscope, if most of the cells to be examined show small "round bubbles", "spots" or "peppercorns", then proceed to step 4 immediately. If most of the cells to be examined show small "round bubbles", "spots" or "peppercorns", the operation in step 4 can be carried out immediately. After 5 min of incubation, if the above situation does not occur, you can also continue to incubate, the time can be as long as 60 min, when the cell surface is small bubbles, step 4 can be carried out in a timely manner.4. Heat the slides at 95°C for 2 min to inactivate proteinase K. Then immerse the slides in PBS for 10 s and in water for 10 s, and then air-dry the slides.5. Add 10 μl of RNAase-free DNA enzyme solution to each sample on the slide, place in a humidified box and incubate at 37°C overnight.
6. Wash the slides once with rinse buffer, then wash them twice with DEPC-treated water and air dry.
7a. If using AMV or MoMuLV reverse transcriptase: Configure the reverse transcription system as follows (total volume 20 μl)
(1) 2 μl 10× AMV/MoMuLV RT buffer (1×)
(2) 2 μl 10 mmol/l mixture of 4 dNTP (final concentration 1 mmol/l)
(3) 0.5 μl 40 U/μl RNasin (final concentration 1 U/μl)
(4) 1.0 μl 20 μmol/l downstream substrate (final concentration 1 μmol/l)
(5) 0.5 μl 20 U/μl AMV or MoMuLV reverse transcriptase (final concentration 0.5 U/μl)
(6) 8 μl DEPC-treated water
7b. If SuperScript 2 reverse transcriptase is used: configure the reverse transcription system as follows (total volume 20 μl)
(1) 4 μl 5× reaction buffer (supplied with enzyme: 1× final concentration)
(2) 2 μl 10 mmol/l 4dNTP mix (final concentration 1 mmol/l)
(3) 0.5 μl 4 U/μl RNasin (final concentration 0.1 U/μl)
(4) 1.0 μl 20 mmol/l downstream primer (final concentration 1 μmol/l)
(5) 0.5 μl 20 U/μl SuperScript 2 (final concentration 0.5 U/μl)
(6) 1.2 μl 0.1 mol/l DTT (final concentration 6 mmol/l)
(7) 4.8 μl DEPC-treated water
8. Add 10 μl of the reverse transcriptase system to each sample on a slide and carefully cover with a 20 mm × 60 mm coverslip, place in a humidified box, and incubate at 42℃ or 37℃ for 1 h.
9. Warm the slide on a 92°C heating block for 2 min, remove the coverslip and wash the slide twice with water.
10. Configure the amplification system as follows (total volume 100 μl)
(1) 5 μl of 25 μmol/l forward primer (final concentration 1.25 μmol/l)
(2) 5 μl of 25 μmol/l reverse primer (final concentration 1.25 μmol/l)
(3) 2.5 μl 10 mmol/l 4dNTP mixture (200 μmol/l final concentration of each dNTP)
(4) 1.0 μl 1.0 mol/l Tris-Cl, pH 8.3 (final concentration 10 mmol/l)
(5) 5.0 μl 1.0 mol/l KCl (final concentration 50 mmol/l)
(6) 2.5 μl 100 mmol/l MgCl2 (final concentration 2.5 mmol/l)
(7) 10 μl 0.01% gelatin (final concentration 0.001%)
(8) 2 μl 5 U/μl Taq DNA polymerase (final concentration 0.1 U/μl)
(9) 66 μl water
Figure I. Lymphocytes before showing proteinase K treatment
11. 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.
12. Place a 20 mm × 60 mm coverslip on each slide and carefully seal the edges of the coverslip with nail polish.13. On a 92°C heating block, warm the slide for 90 s. Then transfer to a thermal cycler.
14. Perform PCR under the following amplification cycle or other optimal conditions(1) 30 cycles: 30 s, 94°C (denaturation)(2) 1 min, about 45°C (annealed)(3) 1 min, 72°C (extension)(4) Final step: indeterminate 4°C (maintenance)15. 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.
Figure 2, showing proteinase K-treated lymphocytes
16. The slides were incubated on a heating block at 92°C for 1 min and then soaked in 2 x SSC for 5 min at room temperature. the slides could be stored at 4°C for 2-3 weeks.