In Situ PCR Amplification of Intracellular mRNA

  • Raymond H. Chen
  • Susan V. Fuggle
Part of the Methods in Molecular Biology™ book series (MIMB, volume 71)

Abstract

The polymerase chain reaction (PCR) is now commonly used in laboratories involved in research studies and clinical diagnostic work (1,2). A major advantage of PCR combined with reverse transcription (RT-PCR) is that it can be used to amplify and detect rare mRNA within a specimen. However, conventional RT-PCR cannot be used either to quantitate the frequency of cells expressing a particular mRNA or determine the cellular origin of the amplified signal. Both of these factors may be relevant in the interpretation of gene expression.

Keywords

MgCl2 Diethyl Hematoxylin Dextran Paraformaldehyde 

1 Introduction

The polymerase chain reaction (PCR) is now commonly used in laboratories involved in research studies and clinical diagnostic work (1,2). A major advantage of PCR combined with reverse transcription (RT-PCR) is that it can be used to amplify and detect rare mRNA within a specimen. However, conventional RT-PCR cannot be used either to quantitate the frequency of cells expressing a particular mRNA or determine the cellular origin of the amplified signal. Both of these factors may be relevant in the interpretation of gene expression.

In order to overcome the limitations of conventional RT-PCR, methods have been developed for performing in situ RT-PCR (for reviews, see 3, 4, 5, 6). By performing the reverse transcription and subsequent amplification within the cells fixed onto microscope slides, it is possible to identify the cellular origin of the signal. The technique has an advantage in that it does not require mRNA to be extracted from the sample, and thus, there is no potential for signal loss during the nucleic acid isolation step. Furthermore, unlike conventional PCR, the technique can be used to determine the prevalence of gene expression within a cell population.

The technique is based on the functional hypothesis that enzymes and reagents can freely enter fixed cells, and synthesize and amplify cDNA in situ. One important corollary is that the PCR products themselves can also freely enter and egress. Consequently, the success of the technique is dependent on an equilibrium between permeability to reagents and the retention of PCR products. In our experience, we found that carefully controlled fixation and digestion were important in maximizing the in situ amplification and retention of signal. The optimization of the equilibrium is critical to the success of the technique, and careful calibration of reaction conditions is mandatory for each primer set used.

In this chapter, we will describe the technique we developed for the detection of granzyme A and perforin mRNA in cytospin preparations of activated human peripheral blood lymphocytes (7). Granzyme A and perforin are functional markers of cytotoxic T-cells, NK, and lymphokine-activated killer cells, and they can be readily detected in adult peripheral lymphocytes after in vitro stimulation (8). Our technique involves directly incorporating biotinylated nucleotides into the PCR product and detecting the labeled product with an antibiotin antibody using a standard immunochemical staining method. This method is not suitable for use on tissue sections, since damaged DNA may serve to prime the reaction, and thus, labeled nucleotides may become incorporated into a nonspecific product (9, 10, 11). Methods for in situ PCR on tissue sections have been described elsewhere (see  Chapters 11 and  12 and refs. 12, 13, 14, 15, 16, 17, 18, 19, 20).

2 Materials

2.1 Glass Slide Preparation

  1. 1.

    Glass slides (Solmedia, Romford, Essex, UK).

     
  2. 2.

    Decon 90 (Decon Laboratories, Hove, UK).

     
  3. 3.

    3-Aminopropyltriethoxysilane (Tespa; Sigma, Poole, Dorset, UK).

     
  4. 4.

    Acetone (Merck, Poole, Dorset, UK).

     
  5. 5.

    Diethyl pyrocarbonate (DEPC; Sigma) treated doubly-distilled water (DEPC-ddH2O; see Note 1)

     
  6. 6.

    Coverslips (Chance Propper, Smethwick, Warley, UK).

     
  7. 7.

    1% Dimethyl dichlorosilane in CCl4 (Merck).

     

2.2 Cell Preparation

  1. 1.

    Anticoagulated human peripheral blood.

     
  2. 2.

    RPMI (Imperial Laboratories, Andover, UK) supplemented with 10% fetal calf serum (FCS; Imperial Laboratories), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin.

     
  3. 3.

    Lymphocyte separation medium (Flow Laboratories, Rickmansworth, Herts., UK).

     
  4. 4.

    Phosphate-buffered saline (PBS; Unipath, Basingstoke, UK).

     
  5. 5.

    Phorbol-12-myristate-13-acetate (PMA, 500 ng/mL; Sigma).

     
  6. 6.

    Phytohemagglutinin (PHA, 1 mg/mL; Wellcome, Dartford, UK).

     
  7. 7.

    Tissue-culture incubator at 37°C.

     
  8. 8.

    Cytospin centrifuge (Shandon Southern Products, Runcorn, Cheshire, UK).

     

2.3 Fixation

Starting from this step, it is imperative that all glassware is baked and all reagents are RNase-free (see Note 2). Chemicals should be reserved for RNA work.
  1. 1.

    4% Paraformaldehyde (Sigma) in DEPC-PBS: Make fresh; heat suspension to 60°C with constant agitation for at least 1 h to allow paraformaldehyde to dissolve in DEPC-PBS. Cool to room temperature before use.

     
  2. 2.

    DEPC-3X PBS, DEPC-1X PBS.

     
  3. 3.

    DEPC-ddH2O.

     
  4. 4.

    50% Ethanol in DEPC-ddH2O, 80% ethanol in DEPC-ddH2O, 100% ethanol.

     

2.4 Proteinase K Digestion

Stock solutions:
  1. 1.

    Proteinase K (Type XI protease [Sigma] 10 mg/mL in 0.1M Tris-HCl, pH 8.0).

     
  2. 2.

    1M Tris-HCl, pH 8.0.

     
  3. 3.

    500 mM EDTA, pH 8.0.

     

2.5 Hybridization and Reverse Transcription

  1. 1.

    Hybridization solution: 50% formamide (Merck), 10% dextran sulfate (Pharmacia, Milton Keynes, Bucks., UK), 300 mM NaCl, 20 mM Tris-HCl, pH 7.6, 5 mM ethylenediaminetetraacetic acid (EDTA), 1X Denhardt’s (Sigma), 10 mM dithiothreitol (DTT, Sigma).

     
  2. 2.

    Antisense primer at 1 µg/mL (see Section 2.6.).

     
  3. 3.

    Moloney murine leukemia virus reverse transcriptase (RT; Life Technologies, Uxbridge, UK).

     
  4. 4.

    5X Reverse transcription buffer (Life Technologies).

     
  5. 5.

    100 mM stocks of dATP, dCTP, dGTP, dTTP (Boehringer Mannheim, Lewes, East Sussex, UK).

     
  6. 6.

    100 mM DTT (Life Technologies).

     
  7. 7.

    RNase inhibitor (Promega, Southampton, UK).

     
  8. 8.

    Bovine serum albumin (BSA [Sigma]) 10 mg/mL in DEPC-H2O.

     
  9. 9.

    DEPC-ddH2O.

     
  10. 10.

    DEPC-2XSSC.

     
  11. 11.

    Humidified slide box.

     
  12. 12.

    Hybridization oven at 42°C.

     
  13. 13.

    Hybridization oven at 37°C.

     

2.6 In Situ PCR

  1. 1.
    Granzyme A primers:
    • 5′-CCA GAA TCT CCA TTG CAC GA

    • 5′-CTG TAA CTT GAA CAA AAG GT

     
  2. 2.
    Perforin primers:
    • 5′-ACA TGG AAA CTG TAG AAG CG

    • 5′-GGA TTC CAG CTC CAT GGC AG

     
  3. 3.

    Taq polymerase (Promega).

     
  4. 4.

    10X Taq polymerase buffer (Promega).

     
  5. 5.

    100 mM stocks of dATP, dCTP, dGTP, dTTP.

     
  6. 6.

    22.5 nmol biotin-11-dUTP (Sigma).

     
  7. 7.

    25 mM MgCl2 (Life Technologies).

     
  8. 8.

    DEPC-ddH2O.

     
  9. 9.

    Mineral oil (Sigma).

     
  10. 10.

    Xylene (Merck).

     
  11. 11.

    Thermocycler (Hybaid, Teddington, Middlesex, UK).

     

2.7 Detection of Amplified Products

  1. 1.

    Mouse antibiotin monoclonal antibody (MAb) (Dako, High Wycombe, Bucks., UK).

     
  2. 2.

    Horseradish peroxidase (HRP)-conjugated, rabbit antimouse Ig antibody (Dako).

     
  3. 3.

    Human AB serum.

     
  4. 4.

    BSA (10% stock solution in PBS).

     
  5. 5.

    Substrate: 3,3-diaminobenzidine tetrahydrochloride (DAB [Sigma], 0.6 mg/mL in PBS made freshly with the addition of 3 µL/mL 3% H2O2 (Thornton and Ross, Huddersfield, UK) immediately before use.

     
  6. 6.

    Harris’ hematoxylin (Sigma).

     
  7. 7.

    70, 90, and 100% Ethanol.

     
  8. 8.

    DPX mountant (Merck).

     
  9. 9.

    PAP pen (Bayer Diagnostics, Basingstoke, Hants., UK).

     
  10. 10.

    Humidified staining tray.

     

3 Methods

3.1 Glass Slide Preparation

  1. 1.

    Glass slides should be thoroughly cleaned by soaking overnight in 10% Decon 90 in double distilled water. Then rinse slides successively with copious amounts of hot tap water, deionized water, and ddH2O, place in racks, dry, wrap in aluminum foil, and bake at 200°C for 4 h to destroy RNase activity. From this point onward, all glassware used should be RNase free (see Note 1). Then coat slides by incubating in a solution of 2% Tespa in acetone for 2 min. Rinse twice with fresh acetone, twice with DEPC-H2O (see Note 1), wrap loosely in aluminum foil, and dry at 37°C overnight. Slides may be stored at room temperature before use.

     
  2. 2.

    Coverslips should be silicon-coated for easy removal during the procedure. Soak the coverslips in 1% dimethyl dichlorosilane in CCl4 for 1 min, rinse with fresh ddH2O, wrap in aluminum foil, bake at 200°C for 4 h, and store at room temperature until required.

     

3.2 Cell Preparation

  1. 1.

    Dilute human peripheral blood 1∶2 with PBS and isolate lymphocytes by centrifugation through lymphocyte-separation medium at 400g at 20°C for 25 min.

     
  2. 2.

    Collect lymphocytes from the gradient interface and dilute at least 1∶2 in PBS; pellet at 400g at 20°C for 7 min.

     
  3. 3.

    Wash twice further in PBS, centrifuging at 300g at 20°C for 5 min.

     
  4. 4.

    Resuspend cells in supplemented RPM1 medium at a concentration of 1 × 106 cells/mL, and stimulate with 100 ng/mL PMA and 50 µg/ mL PHA in RPMI/FCS.

     
  5. 5.

    Four days later, harvest cells by centrifugation at 300g at 20°C for 5 min.

     
  6. 6.

    Wash cells in DEPC-PBS buffer and pellet onto Tespa-treated microscope slides by Cytospin centrifugation at 80g at 20°C for 5 min.

     

3.3 Fixation

  1. 1.

    Place Cytospin preparations on Tespa-treated glass slides in baked slide racks.

     
  2. 2.
    After allowing the slides to dry for 5 min, fix the cytospin preparations at room temperature according to the following schedule (see Note 2):
    1. a.

      4% paraformaldehyde, 20 min;

       
    2. b.

      3X DEPC-PBS, 5 min;

       
    3. c.

      1X DEPC-PBS, 5 min;

       
    4. d.

      1X DEPC-PBS, 5 min;

       
    5. e.

      DEPC-ddH2O, 1 min; 50% EtOH in DEPC-ddH2O, 1 min;

       
    6. f.

      80% EtOH in DEPC-ddH2O, 1 min; and

       
    7. g.

      100% EtOH, 1 min.

       
     
  3. 3.

    After fixation, cytospin preparations may be covered in aluminum foil and stored at −80°C.

     

3.4 Proteinase K Digestion

  1. 1.

    Place slides in a baked 2-L beaker at 37°C for a 30-min digestion with 10 µg/mL proteinase K in 0.11M Tris/50 mM EDTA, pH 8.0 (see Note 2).

     
  2. 2.

    Subsequently fix slides in 4% paraformaldehyde as in Section 3.3.

     

3.5 Hybridization and Reverse Transcription

For all of the manipulations described below, the slides are placed on a bench covered with aluminum foil.

  1. 1.

    Hybridize cytospin preparations with 10 µL of hybridization solution containing 2.5 ng/µL of antisense oligonucleotides to the human granzyme A or perforin genes in a humidified chamber for 2 h at 42°C (see Note 3).

     
  2. 2.

    During this incubation, the preparations are covered with a coverslip, and carefully placed onto the cells using baked forceps.

     
  3. 3.

    At the end of the incubation, wash slides vigorously in 2X SSC for 5 min to remove coverslips and hybridization buffer.

     
  4. 4.

    Shake slides vigorously, wipe with tissue to remove excess salt, and then air-dry.

     
  5. 5.

    Apply 7 µL of reverse transcription mixture to the cell pellet, cover with a coverslip, and incubate for 1 h in a humidified chamber at 37°C.

     
  6. 6.

    The mixture contains reverse transcriptase (3 U/µL), in a buffer of 75 miM KCl, 10 mM Tris, pH 8.0, 12 mM MgCl2, 2 µg/µL BSA, 10 mM DTT containing 1 mM of each dATP, dGTP, dCTP, dTTP, and 1 U/µL RNase inhibitor.

     
  7. 7.

    Wash slides extensively in 2X SSC buffer for 5 min, briefly rinse in ddH2O, and air-dry.

     

3.6 In Situ PCR

Controls are critically important for in situ PCR. (Please refer to Notes 4 and 5 for suggestions.) In order to reduce the quantity of reagents required for the PCR stage, cut coverslips to a size of approx 1 cm2 using a diamond glass cutter.

  1. 1.

    Add 5 µL of a solution containing Taq polymerase at 0.5 U/µL; 1 mM dATP, dGTP, dCTP; 0.9 mM dTTP; 0.1 mM biotin-11-dUTP; 75 mM KCl; 10 mM Tris, pH 8.0; 10 mM MgCl2; and 7 pmol/µL of each 5′ and 3′ oligonucleotide complementary either to the human granzyme A or perforin genes to the slides (see Note 6).

     
  2. 2.

    Place slides on the thermocycler (see Note 7), cover the mixture with the small coverslips, and flood the slides with mineral oil to prevent desiccation (see Note 8).

     
  3. 3.

    The amplification proceeds for one cycle at 94°C for 5 min; 30 cycles at 94°C for 1 min, 60°C for 1 min, 72°C for 1 min; one cycle of 72°C for 10 min.

     
  4. 4.

    After PCR amplification, submerge the slides in xylene for 2 min to remove mineral oil and then leave in the fume hood to allow xylene to evaporate.

     
  5. 5.

    Spray the slides with 70% alcohol and wipe with tissue to remove any remaining oil (see Note 9).

     
  6. 6.

    When dry, place the slides in a rack and agitate in 2X SSC to remove coverslips. Then wash extensively in 2X SSC, in PBS, and finally air-dry.

     

3.7 Detection of Amplification Products

  1. 1.

    Draw a circle around the cell pellet with a PAP pen in order to create a barrier to contain reagents for the detection step.

     
  2. 2.

    The detection step is performed in a humidified chamber.

     
  3. 3.

    Rinse slides in PBS and incubate for 30 min with 50 µL of mouse antibiotin MAb (1∶25 in PBS containing 0.5% BSA).

     
  4. 4.

    Wash the slides three times in PBS, incubating for 5 min each time.

     
  5. 5.

    Detect the primary antibody by incubating for 30 min with 50 µL of HRP-coqugated rabbit antimouse Ig antibody (1∶50 in PBS with 10% human AB serum and 0.5% BSA).

     
  6. 6.

    After washing a further three times in PBS, develop the signal with the freshly prepared substrate solution (DAB and H2O2).

     
  7. 7.

    Counterstam with Harris’ hematoxylin.

     
  8. 8.

    Dehydrate through 70, 90, and 100% ethanol (1 min each), equilibrate in xylene, and mount in DPX medium.

     
Fig. 1.

In situ cDNA PCR detection of granzyme A. (A) Granzyme A was detected in PHA/PMA stimulated lymphocytes following 30 cycles of PCR amplification. (B) PHA/PMA-stimulated lymphocytes were mixed 1∶4 with unstimulated, granzyme A-negative, peripheral blood lymphocytes. Large blast cells are granzyme A-positive, whereas the smaller, unstimulated lymphocytes are negative. This demonstrates that, in this system, despite the presence of labeled PCR product in the supernatant, cells negative for granzyme A within a mixed-cell population remain unstained.

Figure 1 illustrates results obtained by using the RT-PCR technique described in this chapter showing granzyme A mRNA in stimulated peripheral blood lymphocytes.

4 Notes

  1. 1.

    It is important to use RNase-free conditions for this technique. Solutions should be treated with 0.1% DEPC for 12 h at 37°C and autoclaved for 30 min before use. Tris buffers cannot be treated directly with DEPC, but should be made with DEPC-treated, autoclaved ddH2O. Glassware should be rendered RNase-free by covering with aluminum foil and baking for 4 h at 200°C. Gloves should be worn at all times. When it is necessary to place slides on the bench, the bench should be covered with aluminum foil and the slides manipulated with baked forceps.

     
  2. 2.

    Cellular fixation and digestion are critical to the success of this technique. The conditions may vary according to the type of cells used, the size of the PCR product, and the stability of the mRNA. The conditions need to be optimized for each new set of experiments.

     
  3. 3.

    The duration of hybridization needs to be empirically determined. Although a longer hybridization time should favor oligonucleotide-mRNA binding, labile mRNA may degrade, resulting in a truncated cDNA.

     
  4. 4.

    Controls are of crucial importance in in situ PCR systems. Positive controls using primers specific for a housekeeping gene need to be performed on the test cells to demonstrate the presence of mRNA. Cells known to be positive for the gene of interest should be included to demonstrate that the reaction conditions have been optimized. Negative controls are particularly important in a system such as we describe, where labeled nucleotides are directly incorporated into the PCR product. In this respect, a PCR in situ hybridization system has advantages and may be preferred. Some suggested negative control reactions are shown in Table 1. A negative result from reaction (a) will demonstrate the absence of endogenous peroxidase in a sample and from (b) the absence of nonspecific binding of the HRP-conjugated secondary antibody. Reactions (c) and (d) will show that there is neither endogenous biotin in the sample nor a signal following RT alone. Reactions (e) and (f) are particularly important controls; a negative result demonstrates that the signal results from amplification of the cDNA, and not from priming by nicked DNA or amplification of genomic DNA. Reaction (g) demonstrates the requirement for primers and (h) on Taq polymerase to produce a signal.

     
  5. 5.

    It is possible to perform a Southern blot analysis on the supernatant from the in situ PCR reaction (3). This enables the specificity of the amplified product to be confirmed.

     
  6. 6.

    In situ PCR appears to require higher concentrations of reagents than tube PCR (MgCl2, Taq polymerase, and nucleotides). Consequently, it may not be possible to transfer conditions optimized for tube PCR directly to an in situ PCR system.

     
  7. 7.

    The heating blocks of traditional PCR thermocyclers, with their discontinuous surface area, do not provide ideal heat conduction for in situ PCR. It is preferable to use a machine with specifically designed flat blocks.

     
  8. 8.

    Desiccation during the PCR process can yield false-positive signals. It is important that the mineral oil completely seal the coverslip.

     
  9. 9.

    The mineral oil should be completely removed before the immunohistochemistry step. If present, the hydrophobic oil droplets will interfere with the antibody binding and subsequent detection of the PCR product.

     
Table 1

Suggested Negative Controls for In Situ cDNA PCR

 

a

b

c

d

e

f

g

h

Fixation, digestion

+

+

+

+

+

+

+

+

Hybridization

+

+

+

+

+

+

+

Reverse transcription

+

+

+

+

+

+ b

+

+

PCR

+

+

+ a

+

+

+ c

+ d

Antibiotin antibody

+

+

+

+

+

+

HRP-conjugated secondary antibody e

+

+

+

+

+

+

+

Substrate

+

+

+

+

+

+

+

+

Expected result

a Omit biotinylated nucleotide.

b Omit reverse transcriptase.

c Omit primers.

d Omit Taq polymerase.

e HRP-horseradish peroxidase.

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Copyright information

© Humana Press Inc, Totowa, NJ 1997

Authors and Affiliations

  • Raymond H. Chen
    • 1
  • Susan V. Fuggle
    • 1
  1. 1.Nuffield Department of SurgeryJohn Radcliffe HospitalHeadingtonUK

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