Molecular Techniques and Methods

In situ Polymerase Chain Reaction
(In situ PCR)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Detection of low-copy DNA in situ has become routine with the widespread use of PCR. With the hot start maneuver, one can routinely detect with PCR one copy in a background of 1 ug of total cellular DNA.




MATERIALS AND SOLUTIONS

Silane-coated Slides
Coat the slides with Silane (Sigma, M6514).


Pepsin Digestion Solution (10 ml)
Pepsin ------------------------------------------ 20 mg
DEPC-treated H2O ----------------------------- 9.5 ml
2 N HCl ---------------------------------------- 0.5 ml


10 mM dNTP Mix (1 ml)
10 mM dATP ----------------------------------- 100 ul of 100 mM dATP
10 mM dCTP ----------------------------------- 100 ul of 100 mM dCTP
10 mM dGTP ----------------------------------- 100 ul of 100 mM dGTP
10 mM dTTP ----------------------------------- 100 ul of 100 mM dTTP
DEPC-treated H2O ----------------------------- 600 ul


Digoxigenin dUTP (Boehringer Mannheim)


Wash Solution (10 ml)
l x SSC ----------------------------------------- 0.5 ml of 20 x SSC
0.2% BSA -------------------------------------- 2 ml of 1% BSA
DEPC-treated H2O ----------------------------- 7.5 ml


Antibody Solution (1 ml)
0.1 M Tris-HCl (pH 7.5) ------------------------ 100 ul of 1 M Tris-HCl
0.1 M NaCl ------------------------------------- 20 ul of 5 M NaCl
DEPC-treated H2O ----------------------------- 880 ul
Anti-Digoxigenin Antibody (1:200 dilution) (Boehringer Mannheim)


Color Development Solution (1 ml)
0.1 M Tris-HCl (pH 9.5) ------------------------ 100 ul of 1 M Tris-HCl
0.1 M NaCl ------------------------------------- 20 ul of 5 M NaCl
DEPC-treated H2O ----------------------------- 880 ul


Antidigoxigenin Conjugate (Boehringer Mannheim)




PROCEDURES

Preparation of Tissue Sections and Cell Samples

1. Fix the tissue samples in 10% Buffered Formalin (Sigma, HT50-1) for 10-15 hours, and then embed in paraffin.

For cell cultures, wash the cells once with PBS directly in the culture plate.
  • Add 10% Buffered Formalin (Sigma, HT50-1), let stand overnight and then scrape the cells off the plate with a rubber policeman.
  • Wash the cells in DEPC-treated H2O twice, and centrifuge at 2,000 rpm for 3 minutes.
  • Resuspend the cells in 5 ml of DEPC-treated H2O, and spot 50 ul on a silane-coated slide.

    2. Place several 4-um paraffin embedded-tissue sections or three cell suspensions on silane-coated slides.
  • The silane coating is essential for cell adherence during the procedure.

    3. Remove the paraffin from the tissue samples by placing the slides in fresh xylene for 5 minutes and then in 100% ethanol for 5 minutes. Air-dry.


    Protease Digestion

    4. Incubate slides in a Pepsin Digestion Solution for 10-40 min at room temperature.
  • For in situ PCR (with direct incorporation of the reporter molecule during PCR), the proper protease digestion time is highly dependent on the time of fixation in formalin.

    5. Inactivate the protease by washing the slide in DEPC-treated H2O for 1 minute and then in 100% ethanol for 1 minute; air-dry.
  • This simple wash step is sufficient to remove/inactivate the protease; heat-inactivation is not required.


    PCR Reaction

    6. Make PCR master mix as follow (for 25 slides).
    >
    Components
    Volume
    Final Concentration
    10 x PCR Buffer
    50 ul
    1 x
    25 mM MgCl2
    90 ul
    4.5 mM
    10 mM dNTP Mix
    10 ul
    200 uM
    2% BSA
    (Bovine Serum Albumin)
    20 ul
    0.08%
    1 mM Digoxigenin dUTP
    8 ul
    16 uM
    20 uM Primer 1
    25 ul
    1 uM
    20 uM Primer 2
    25 ul
    1 uM
    DEPC-treated H2O
    262 ul
    -
    Taq DNA polymerase
    15-20 U
    -
    Final Volume
    500 ul
    100 ul


    7. Add 25 ul In situ PCR Reaction Buffer to each slide and cover with one large coverslip.
  • Anchor the coverslip with two small drops of nail polish.

    8. Do PCR reaction as follow.

    1 cycle
    Denaturation
    95oC
    3 min
    13-17 cycles
    Denaturation
    95oC
    1 min
    Annealing and Extension
    Tm-5oC
    (>55oC)
    2 min


    9. Remove the coverslip and polish.
  • Wash for 5 minutes in xylene.
  • Wash for 5 minutes in 100% ethanol.
  • Air-dry slides.


    Signal Detection

    10. Wash the slides for 3 minutes in Wash Solution at 52oC.

    11. Remove the excess Wash Solution.
  • Add to each slide 100 ul of Antibody Solution.

    12. Incubate at 37oC for 30 minutes.

    13. Wash the slides for 1 minute in the Color Development Solution.

    14. Then incubate the slides in the Color Development Solution with NBT/BCIP chromagen.
  • Incubate at 37oC for 10-60 minutes.

    15. Check the slides under a microscope and stop the reaction when the signal is strong.

    16. Wash the slides in water for 1 minute.

    17. Counterstain the slides with nuclear fast red for 5 minutes.

    18. Wash the slides in water for 1 minute.
  • Wash the slides in 100% ethanol for 1 minute.
  • Wash the slides in xylene for 1 minute.

    19. Mount coverslip using Permount.
  • View under a microscope.




    NOTES

    Preparation of Tissue - The choice of fixative is essential for in situ PCR. Successful in situ hybridization and PCR can be done with unfixed, frozen tissue or after fixation in acetone, ethanol, or buffered formalin.

    10% Buffered Formalin (Sigma, HT50-1) Cross-link proteins and nucleic acids and has a mode of action very different from acetone and ethanol. Formalin fixation appears to create a migration barrier that, under certain specified conditions, severely limits the movement of the PCR product from its site of synthesis in the cell. It is reasonable to assume that this relates to the protein-DNA latticework that occurs secondary to the cross-links created after formalin fixation.
    Acetone and Ethanol Denature proteins and, in this way, render degradative enzymes inoperative. Ethanol and acetone fixation allow intranuclear DNA synthesis, but that, in many cells, the PCR product migrates out of the cell.
    10% Buffered Formalin (Sigma, HT50-1) with Picric acid (e.g., Bouin's solution) or with a Heavy Metal such as Mercury (e.g., Zenker's solution) Give better nuclear detail with microscopic sections (bone marrow and lymph node biopsies). However, fixatives that include a heavy metal or picric acid do not allow PCR because of the rapid and extensive degradation of the DNA. Tissues fixed for more than 8 hours in solutions that contain either a heavy metal or picric acid do not permit a signal with in situ hybridization, although shorter-term fixation can yield intense signals.
    Frozen, Unfixed Tissues For preservation of the antigens determinant


    Protease Digestion - The signal evident with in situ PCR for formalin-fixed tissues is strongly dependent on the length of time for both formalin fixation and digestion with protease. It is very important to stress that these data apply to direct incorporation of the reporter molecule into the PCR product (in situ PCR). This is to be differentiated from PCR in situ hybridization, where the PCR product is detected by a labeled internal oligoprobe after a hybridization step. The optimal protease digestion time does not vary much with PCR in situ hybridization, where it is from 20 to 30 minutes for tissues that have been fixed for 4 hours to several days.

  • Most proteases (e.g. proteinase K, pepsin, trypsin, trypsinogen, and pronase) allow successful in situ amplification of DNA and cDNA. We prefer trypsin or pepsin at a low pH, because a simple change of pH to 7 is enough to reduce the activity of the protease. Also, proteinase K tends to cause overdigestion more commonly. The optimal protease time is dependent on the time of formalin fixation. Inadequate protease digestion is the most common cause of unsuccessful in situ PCR.

  • If the negative control shows positive cells, then increase the protease digestion time. If both the negative and positive controls do not show positive cells, then check the tissue morphology. If it is poorly preserved, decrease the protease digestion time. If it is well preserved, increase the protease digestion time.


    Solutions for PCR Amplification - An optimal signal for in situ PCR was obtained with 4.5 mM of MgCl2. This relatively high and constant concentration of magnesium probably reflects its partial sequestration on the glass slide and cellular proteins.

  • BSA is commonly used in immunohistochemistry to inhibit nonspecific binding of proteins to the slide or tissue section. The addition of BSA allowed an intense hybridization signal after in situ PCR with a less Taq DNA polymerase concentration of 1.5 units per 50 ul.


    Nonspecific Amplification during In situ PCR - Primer-independent DNA synthesis occurs if paraffin-embedded fixed tissues are used for in situ PCR. This signal results from the formation of DNA nicks during the 65oC paraffin-embedding process. Because of this background, direct incorporation of the reporter group into the product is not recommended for formalin-fixed, paraffin-embedded tissues for DNA targets. Instead of in situ PCR with labeled nucleotides, we recommend the PCR in situ hybridization.




    KIT INFORMATION

    Many reagents, including the protease, buffers, washes, chromagen, probes, and counterstains, are part of comprehensive in situ hybridization kits, which are now marketed by several biotechnology companies.




    REFERENCES

  • Crum, CP, Nuovo, GJ, Friedman, D, Silverstein, SJ (1988) A comparison of biotin and isotope labeled ribonucleic acid probes for in situ detection of HPV 16 ribonucleic acid in genital precancers. Lab. Invest. 58: 354-559.

  • Greer, CE, Peterson, LS, Kivian, NB, Manos, MM (1991) PCR amplification from paraffin-embedded tissues-Effects of fixative and fixative times. Am. J. Clin. Pathol. 95: 117-124.

  • Nuovo, GJ, Silverstein, SJ (1988) Comparison of formalin, buffered formalin, and Bouin's fixation on the detection of human papillomavirus DNA from genital lesions. Lab. Invest. 59: 720-724.

  • Nuovo, GJ, Gallery, F, MacConnell, P, Becker, J, Bloch, W (1991) An improved technique for the detection of DNA by in situ hybridization after PCR-amplification. Am J. Pathol. 159: 1239-1244.

  • Nuovo, GJ (1994) PCR in situ hybridization: Protocols and Applications, 2nd edition. Raven Press, New York.



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