In Situ RT-PCR and Hybridization Techniques

Abstract

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ability to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1–10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13).

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1 Introduction

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ability to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1–10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13).

The recent introduction of new enzymes (14,15), sealing reagent, and automatic in situ PCR machines have made this technique much easier and less time-consuming. This technique has been improved enormously in the past two years. It is possible to obtain reproducible results under carefully designed reaction conditions with the proper selection of reagents and equipment. In situ PCR technology has a tremendous potential for its applications in diagnostic histopathology, viral diseases, study of in situ gene expression, regulation, and mutation (16–21).

The following protocol and discussion contains fundamental principles and as much detail as possible but remains a general outline of the procedures and practical considerations. Each individual experiment must be well planned, with sufficient theoretic contemplation given to the unique characteristics of the study target and experimental materials. Whenever possible, practical procedural tips have been included in an attempt to save time, trouble, and materials.

There are two different approaches to performing the in situ reverse transcription (RT)-PCR technique (22–25).

• 1. Direct in situ RT-PCR: Direct detection of the amplified products by incorporating digoxigenin-labeled 11-dUTPs during the PCR reaction, followed by binding with the Fab fragment enzyme-conjugated (alkaline phosphatase) antidigoxigenin antibody and subsequent staining of the complex with the specific substrate Nitroblue-tetrazolium and 4-bromo-5-chloro-3-indolylphosphate (NBT-BCIP).

• 2. Indirect in situ RT-PCR: Indirect detection of the PCR amplified signal by hybridization with a digoxigenin-labeled oligo probe specific for the target followed by binding with the alkaline phosphatase-conjugated antidigoxigenin antibody and detection of the hybridized complex by staining with the specific substrates (NBT-BCIP).

2 Materials

2.1 Equipment and Glassware

• 1. Microtome.

• 2. 65°C oven.

• 3. In situ PCR machine (Thermal Cycler PTC-100™M. J. Research, Watertown, MA).

• 4. Microcentrifuge.

• 5. Benchtop centrifuge.

• 6. Vortex machine.

• 7. pH meter.

• 8. Magnetic stirrer.

• 9. Hybridization oven.

• 10. A chemical hood.

• 11. A laminar flow or a tissue culture hood.

• 12. UV chamber or DNA station (M. J. Research).

• 13. Incubator.

• 14. Water bath.

• 15. Refrigerator.

• 16. Freezer.

• 17. A light microscope.

• 18. Autoclave and a baking oven for glassware.

• 19. Humidity chamber (Shandon Lipshaw, Pittsburgh, PA; or to make your own, see Note 1 ).

• 20. Thermometer.

• 21. Forceps, scalpels, labeling tapes.

• 22. Slide holders.

• 23. Glass and plastic Coplin jars or staining dishes (Fisher Scientific, Pittsburgh, PA).

• 24. 20 × 30-mm glass cover slips (Bellco Glass, Co. Vineland, NJ).

• 25. Plastic cover slips (PGC Scientific, Gaithersburg, MD).

• 26. Silanated slides (Digene Diagnostics, MD).

• 27. Disposable sterile polypropylene tubes (15- and 50-mL capacity).

• 28. RNase-free Eppendorf tubes (see Note 2 ).

• 29. Micropipetters.

• 30. Microtips (range 0.5–1000 μL).

• 31. Tube racks.

• 32. Measuring cylinders.

• 33. Funnels.

• 34. Ice buckets.

• 35. Slide holders.

• 36. Aluminum foil.

2.2 Reagents

• 1. Diethyl pyrocarbonate (DEPC) (Fluka Chemical Corp., Ronkonkoma, NY).

• 2. DEPC-treated water: 800 μL DEPC/L of distilled water; stir overnight or at least 1 h; autoclave (see Note 3 ).

• 3. 10% buffered formalin.

• 4. 4% paraformaldehyde (Fluka): heat 400 mL DEPC-treated 1X PBS to 65°C; add 16 g paraformaldehyde; stir with a magnetic stirrer until it dissolves (approx 1–2 h); adjust pH to 7.5 with 10 N NaOH (see Note 4 ).

• 5. RNASE Zap (Ambion, Inc., Austin, TX).

• 6. Xylene.

• 7. Absolute alcohol.

• 8. Graded alcohols: 50% ethanol: mix DEPC-water and absolute ethanol 1:1; 70% ethanol: mix DEPC-water and absolute ethanol 1:3; 95% ethanol: mix DEPC-water and absolute ethanol 5:95.

• 9. DEPC-treated PBS (10X, 3X, 1X); for 1L DEPC-treated 10X PBS: 23.5 g Na₂HPO₄, 4.5 g NaH₂PO₄, 87.6 g NaCl, 0.8 mL DEPC solution, 1000 mL dH₂O; autoclave for 20 min. Dilute with DEPC treated-dH₂O to make 3X and 1X.

• 10. UV-irradiated double-distilled water (UV-ddH₂O) (see Note 5 ).

• 11. Proteinase K (Fluka) or Pepsin (Boehringer Mannheim Biochemicals, Indianapolis, IN); for stock solution: 10 mg Proteinase K, 10 mL 1X DEPC-treated PBS; mix and store 1-mL aliquots in RNase-free Eppendorf tubes at -20°C.

• 12. 0.1 M glycine in 1X PBS: mix 7.5 g glycine/L of 1X DEPC-treated PBS.

• 13. 0.3% hydrogen peroxide (H₂O₂) solution in methanol: 1.0 mL 30% H₂O₂ and 99.0 mL absolute methanol.

• 14. Methanol.

• 15. 0.02 N HCl: mix 0.2 mL 12 N HCl and 119.8 mL DEPC-dH₂O (see Note 11 ).

• 16. Reverse transcription reagents (Life Technologies, Inc., Germantown, MD): antisense primer; ultrapure 4(dNTPs), 100 mM each; dithiothreitol (DTT); oligo dT; first-strand reaction buffer, 50 mM Tris-HCl, pH 8.3, 5 mM KCl, 3 mM MgCl₂; SupersciptII (Reverse Transcriptase); RNasin (Promega, Madison, WI); UV-irradiated dH₂O.

• 17. PCR reaction reagents (Perkin-Elmer and Life Technologies, Inc., Foster City, CA): Ultrapure 100 mM4(dNTP) stock solution; UV-irradiated dH₂O; Digoxigenin 11-dUTP (Boehringer Mannheim); PCR cocktail, 10X PCR buffer, MgCl₂, a set of sense and antisense primers, BSA, Taq polymerase-Taq-start antibody complex (see Note 6 ).

• 18. Taq start antibody (CLONTECH Laboratories, Palo Alto, CA).

• 19. Digoxigenin-11-ddUTP (Boehringer Mannheim).

• 20. Hybridization solution (Boehringer Mannheim): Deionized formamide; 20X SSC; 100X Denhardt solution; 10 mg/mL Salmon sperm DNA; 10% SDS.

• 21. 2X SSC: 300 mM NaCl, 30 mM sodium citrate.

• 22. Detection buffers: 1 L Buffer 1: combine 12.11 g Tris; 5.84 g NaCl; 0.40 g MgCl₂; 30.0 g BSA; adjust to pH 7.5. 1 L Buffer 2: combine 12.11 g Tris; 5.84 g NaCl, 10.0 g MgCl₂; adjust to pH 9.5. 1 L Buffer 3: combine 2.42 g Tris; 1.86 g EDTA; adjust to pH 7.5.

• 23. Anti-digoxigenin antibody, a Fab fragment (Boehringer Mannheim).

• 24. 0.1 M Tris-HCl, pH 7.5: Combine 12.11 g Tris/L of DEPC-water and adjust the pH with pure concentrated 12 N HCl.

• 25. 0.1 M Tris/50 mM EDTA, pH 8: Combine 12.11 g Tris, 18.61 g EDTA/L of DEPC-water and adjust pH.

• 26. Nitroblue tetrazolium (NBT).

• 27. 4-bromo-5-chloro-3-indolylphosphate (BCIP).

• 28. Mounting medium (Aqua mount, Biomeda c/o Fisher Scientific, Inc., Pittsburgh, PA) (see Note 17 ).

3 Overview of Protocol and Fundamental Principles (see Notes 18 and 19 )

3.1 Fixation and Sample Preparation

Experimental samples are mainly derived from tissue culture cells, laboratory animals, or human tissues collected from hospitals after surgical biopsies and autopsies. With human and animal tissue specimens, it is important to arrest metabolic processes within 5–10 min of collection in order to preserve mRNAs from degradation by internal enzymatic reactions (26,27). Most hospitals use 10% buffered formalin as a tissue fixative. Subsequently, each tissue slice is trapped in a paraffin block. Series of 4–5-μm-thick sections are cut and mounted on silanated slides. Formalin-fixed archival tissues have been successfully used in in situ PCR and in situ hybridization protocols (28–32). However, the procedure for RNA protection is not always followed. It is often difficult to alter or control the routine procedures of hospitals for the required protection of mRNAs in surgically removed human tissues.

Proper fixation is one of the most critical steps in an in situ RT-PCR or in situ hybridization experiment, because each tissue type must have optimized fixation conditions. Particularly archival tissues may require individual specific treatment in order to meet with in situ experimental requirements. Errors in fixation will only be discovered after the entire hybridization process has been completed (see Note 10 ).

When selecting the appropriate fixative, its possible effect on tissue morphology, target signal retention, and the PCR process (with particular regards to temperature sensitivity) must be carefully considered. You may choose between crosslinking or precipitating fixative types according to the tissue sample being used. You will know that you have achieved the best fixative results when the probe and reagents have sufficiently penetrated your tissue sample and have provided strong signal results, yet all of the target DNA or RNA has been retained and the morphology of the tissue sample is intact (33) (see Note 11 ).

Although the best probe penetration might be achieved with precipitating fixatives such as acetic acid or ethanol, imperfect tissue morphology and a loss of target signal may also result. Improved RNA retention and tissue morphology can be achieved with the use of aldehyde (cross-linking) fixatives and using a 4% paraformaldehyde solution is a common compromise (34). The proper balance between target retention and tissue permeability must be determined by trial and error according to the target selected. Consideration should be given to the fact that mRNA is degraded enzymatically, whereas DNA is more stable. RNA studies require that the tissue be fixed or frozen within 10 min of collection in order to retain sufficient message, and the time elapsed between sample collection and fixation should always be accounted for when the results are interpreted.

The in situ RT-PCR process subjects tissue samples to various severe chemical and enzymatic reactions and temperature fluctuations. For that reason, silanated or positively charged slides must be used that are able to retain the tissue sample throughout the process.

Tissue culture cells are easy to handle under laboratory conditions. It is necessary to fix these cells immediately after cytospin or before paraffin embedding. Different fixatives are used depending on the goal of the experiment.

One should avoid the use of highly cross-linking fixatives, such as mercuric chloride, glutaraldehyde, modified formalin, and picric-acid-based fixatives. These extensively cross-linking fixatives render the tissue virtually impermeable to the RT-PCR reaction components and to the probe.

3.1.1 Preparation of Paraffin-Embedded Tissue Culture Cell Sections

For in situ mRNA detection:

• 1. Grow cells in five T-150 tissue culture flasks cells either in suspension or in monolayer.

• 2. When growth reaches 60–70% confluency, harvest cells by trypsinizing and spinning the cells at 500g in 15-mL centrifuge tube for 10 min.

• 3. Wash the cell pellet 3X with DEPC-treated 1X PBS.

• 4. After carefully aspirating the supernatant, add 10% buffered formalin or fix it in freshly prepared 4% paraformaldehyde for 4–16 h. After this step follow the standard guidelines for paraffin-wax embedding as described in Subheading 3.1.2.

3.1.2 Paraffin-Wax Embedding

• 1. Place tissues from laboratory animals or surgically removed human tissue samples in freshly prepared 4% paraformaldehyde for 4–16 h at room temperature.

• 2. Transfer the tissues in 0.5M sucrose in 1X PBS at room temperature for 8–12 h.

• 3. Process in the following:

  • a. Normal saline: (0.85% NaCl), 15 min, two times.

  • b. Normal saline: ethanol (1:1), 30 min, two times.

  • c. Ethanol 70%, 30 min, two times.

  • d. Ethanol 85%, 30 min, one time.

  • e. Ethanol 95%, 30 min, one time.

  • f. Ethanol 100%, 30 min, two times.

  • g. Xylene, 20 min, three times.

  • h. Xylene: paraffin (1:1), 20 min, two times,

  • i. Paraffin, 20 min, three times.

• 4. Embed tissue samples in paraffin wax in embedding cassettes.

• 5. Place the embedded tissues at 4°C for 2–12 h.

3.2 Preparation of Positive and Negative Controls

For positive controls, it is extremely important to have a tissue culture cell line with an abundant amount of target nucleic acid. Varieties of tissue culture cell lines are available from American Tissue Culture Center, Rockville, MD. Rapidly growing tissue culture cell lines transfected with the target nucleic acid could be successfully used for positive controls. For example, formalin fixed paraffin-embedded foreskin fibroblast tissue culture cell-line FS4 can be used as a positive control for human lysyl oxidase mRNA detection and c-H-ras transformed RS-485 cell line for normal ras message detection. Thin sections (4–5 μm) of paraffin-embedded positive control cell lines could be placed simultaneously near the side of the experimental human or animal tissue. Similarly, one can also use freshly cut animal or human tissue (with the target sequences) preserved, sectioned, and mounted under standard laboratory conditions. For negative controls, choose a cell line (or a tissue) that completely lacks the target nucleic acid (see Note 12 ).

3.3 Glass Slide Preparation

While performing the protocol of in situ RT-PCR on the tissue sections, each tissue undergoes a variety of relatively harsh chemical and enzymatic treatments and temperature shocks. Hence, the tissue or cells should be mounted on slides that can best retain and hold the specimen under relatively harsh chemical and temperature treatment.

Varieties of pretreated precoated slides are commercially available. Your choice of coating on the slide can play an important role. I started my work with Teflon-coated three-well slides but soon realized that the area available for mounting the tissue was much smaller than the tissue sizes from the human tissue blocks. It was also very time-consuming and inconvenient to seal three-well Teflon-coated slides with a clear nail polish. I personally use commercially made silanated slides from Digene Diagnostics, Silver Spring, MD. As an alternative, Dr. Bagasra’s protocol for silanating plain slides works very well (35).

3.3.1 Specimen Mounting

• 1. Cut formalin- or paraformaldehyde-fixed paraffin-embedded tissue into 4–5-μm-thick sections on a microtome.

• 2. These tissue sections are floated in a nuclease-free clean waterbath filled with DEPC-treated water.

• 3. Place each section on a silanated slide by scooping the slide under the floating tissue section and lifting the slide with the attached tissue section up and out of the waterbath.

• 4. With a soft paint brush gently remove water bubbles and foldings of the tissue. After mounting sections to the slides, allow to air-dry.

• 5. The day before the experiment, bake slides at 65°C overnight.

3.4 Pretreatment/Proteinase K Digestion

Tissues fixed with crosslinking fixatives require extensive pretreatment to permeabilize the tissue in order to allow the reverse transcription and polymerase chain reaction components access to the target nucleic acid sequences. The success of the experiment depends on the careful optimization of permeabilization and proteinase K digestion (36). Overdigestion with proteinase K can cause breakage in the tissue morphology, and tissues might become fragmented and partially or completely fall off the slide. Excessive pretreatment can also cause a leakage of the amplicon (PCR-amplified product) into other areas of the tissue, causing ambiguous results (1,27).

• 1. Refix the tissue in freshly prepared 4% paraformaldehyde for 1–4 h after xylene treatment and before proteinase K digestion (see Note 13 ).

• 2. For optimizing pretreatment conditions, treat a series of tissues (sectioned from the same tissue block) with different concentrations of proteinase K (5, 10, 15, 20, 25, 30 μg/mL) and incubate for 15 min at 37°C in the humidity chamber.

• 3. At the end of incubation, observe the integrity of the tissue morphology under the light microscope. One should choose the highest concentration of proteinase K that can retain the intact tissue morphology. Overdigested tissues that show distortion, fragmentation, wavy texture, or a loss of tissue morphology because of partial detachment of the tissue from the slide should be discarded (see Note 14 ).

3.5 DNase Digestion

It is necessary to treat the tissues with highly pure RNase-free DNase to destroy all the endogenous DNA in the cells and tissues so that only RNA remains available for cDNA synthesis and amplification. This avoids problems associated with the incorporation of labeled nucleotides into DNA through repair mechanism during the reaction cycles of in situ RT-PCR and prevents nonspecific amplification of DNA gene sequences by Taq DNA polymerase (37).

3.6 Primer Design

A set of sense and antisense primers should be selected to synthesize specific cDNAs and also to detect the amplified messages of the genes, complementary to their specific gene sequences. It is important to consider the following points while designing the primers for reverse transcriptase and polymerase chain reaction:

• 1. Length of sense and antisense strands should be between 20 and 30 bp.

• 2. At the 3′ ends of both primers, there should be at least one CG-type basepair in any combination to facilitate complementary strand formation.

• 3. The GC content of the primers should be between 45 and 55%.

• 4. 3′ ends of primers should not be complementary to avoid primer dimer formation.

• 5. Reverse transcription primers should be designed so that they do not contain secondary structures (38).

3.7 Reverse Transcription Reaction (cDNA Synthesis)

In the cells and tissues, low- or single-copy number viral RNA or messenger RNA can be detected by in situ RT-PCR. Templates of mRNAs are transcribed to the first strand complementary DNAs (cDNAs) by incubating the tissue sections with the specific RT-reaction mixture. A variety of reverse transcriptase enzymes are available commercially. The first strand synthesis protocol varies depending on the type of RT enzyme and its specific reaction conditions. One should carefully follow the instructions recommended by the enzyme manufacturing companies. The optimum temperature for the first strand cDNA synthesis ranges between 42 and 55°C (39). The cDNA is synthesized by reverse transcriptase in the reaction mix containing either antisense highly specific primer or random hexamers and free nucleotides (dNTPs), at approx 42°–50°C for approx 60 min. Subsequent heating at 70°C for 10 min and rapid cooling for 1 min keeps the cDNA products in a linearized form. The newly formed cDNA becomes the template for the polymerase chain reaction (see Notes15 and 16 ).

3.8 The Polymerase Chain Reaction

The cDNAs, which are assumed to be synthesized at the end of the RT reaction, are subjected to amplification reaction by following two methods. The first, “Indirect in situ PCR” method, involves the incorporation of unlabeled nucleotides (40). In the indirect method, the target cDNA is amplified by unlabeled nucleotides. This is immediately followed by the hybridization step with the digoxigenin-labeled oligo probe. The resulting digoxigenin-labeled hybridized product is then detected by using the target detection method. In the second, “Direct in situ PCR” method, digoxigenin-labeled 11-dUTPs are added into the cocktail of the regular dNTPs, and their ratio is maintained as recommended by the manufacturers (Boehringer Mannheim). This amplification does not require the subsequent hybridization step by labeled probe (41). The digoxigenin-labeled amplified target can be directly detected by following the signal detection protocol. This protocol is relatively short and easy to perform. However, it takes a long time to optimize reaction conditions. There are more chances to get false-positive signals by the repair processes of nicked and damaged DNA. It is also less specific because it lacks the step of subsequent hybridization of the amplicon with a highly specific labeled probe (42,43).

3.8.1 The Hot-Start Strategy

Initial annealing between the primers and the target sequence determines the amplification specificity. “Hot start” entails the initiation of a primer-target annealing step at a higher temperature, which significantly reduces the possibility of mispriming and thereby improves the specificity of subsequent PCR (44). Under the in situ conditions, the “hot-start” could be performed by adding into the PCR reaction system either the enzyme AmpliTaq Gold (specially designed for “hot start” Perkin-Elmer) or the complex of Taq polymerase enzyme-Taqstart™ antibody (PT1576-1, CLONTECH). Each step of the protocol should be handled carefully (see Notes 17 and 18 ).

3.9 Hybridization

For indirect detection of unlabeled RT-PCR products, slides are hybridized with a digoxigenin-labeled probe. The probe should be chosen so that it spans an exon-intron junction, ensuring that the hybridization occurs only with messagederived products and not with fragments of genomic DNA. Synthetic oligo probe detects only one of the two amplified target cDNA sequences. I have used 3′ end digoxigenin-labeled synthetic oligonucleotide probe in my indirect in situ RT-PCR experiments.

3.9.1 Preparation of the Probe

In the tissue sections, a hybridization is performed with a highly specific probe in order to detect the target (either amplified by indirect in situ RT-PCR or unamplified). For the preparation of nonradioactive digoxigenin-labeled probes, one should refer to the guidelines of the company manuals: Genius™ System User’s guide for membrane hybridization and Nonradioactive in situ hybridization application manual (2nd ed., Boehringer Mannheim).

For labeling synthetic oligonucleotides with digoxigenin, there are three methods developed by Boehringer Mannheim: 3′ end labeling, 3′ end tailing, and 5′ end labeling. The PCR-generated digoxigenin-labeled probes are also being successfully used in hybridization-detection methods. I have used both 3′ end labeled synthetic oligonucleotide probe (24 bp) as well as PCR-generated double-stranded cDNA probe (238 bp) to detect lysyl oxidase message in human prostate tissues. The gel- or HPLC-purified oligonucleotide probe is labeled at the 3′ terminus with digoxigenin-11-ddUTP, using terminal transferase, but only one molecule of digoxigenin becomes attached. However, it is highly specific, and sometimes the signal develops after 12–24 h of incubation with the substrate. The hybridization with PCR-generated double-stranded cDNA probes (usually 200–300 bp) provides a higher concentration of digoxigenin to bind with the enzyme (alkaline phosphatase, horseradish peroxidase) or fluorescein (FITC)-conjugated antidigoxigenin antibodies. The intensified signal can be detected within 10 min to 2 h.

3.9.2 Posthybridization

After the hybridization step, the posthybridization wash conditions should be optimized for each type of probe, whereby an optimized stringent posthybridization wash should be balanced so that the much weaker and fewer hydrogen bonds between the probe and nontarget molecules are disrupted but enough below the melting point temperature (T _m) of the probe/target complex (1). Under low-stringency wash conditions, oligo probes tend to produce more background than larger PCR-generated cDNA probes or genomic probes. Another difference between oligo probes and larger genomic probes relates to the actual conditions of the posthybridization wash. It is easy to achieve a temperature with larger probes for the posthybridization wash in which the specific signal persists and the background signal is lost. However, these conditions could be too stringent with the oligo probe. One must reduce the stringency until one reaches the narrow window above the Tm of the background hybridization yet below the T _m for the oligo probe-target annealing (see Note 19 ).

3.10 Signal Detection

Indirect in situ RT-PCR protocol requires a hybridization step for the detection of the amplified target cDNA products. Boehringer Mannheim color detection kits or separate detection reagents are available for direct or indirect detection of digoxigenin-labeled cDNA targets. The sample must first be blocked using blocking agent (either BSA or a sheep serum) to block any protein binding sites to which the antibody conjugate may bind. This is followed by incubation of a tissue sample using alkaline phosphatase- or horseradish peroxidase-conjugated antidigoxigenin antibodies. A freshly prepared substrate solution with NBT and BCIP in alkaline buffer solution is then added on the top of each tissue, and the chamber is placed in the dark for the development of the purple-blue-colored signal. The reaction is stopped by transferring the slides into a solution containing Tris-EDTA after observing under the microscope for the proper intensity of the colored signal. Excessive background can be removed by dipping the slides for 5–7 min in 95% methanol (see Notes 20 and 39-1). Finally, the slides are mounted with a mounting media (Aquamount/Crystal mount, Biomeda) (see Notes 17 , 22 , and 23 ).

4 Method

For normal and neoplastic human breast and prostate tissues.

4.1 Day 0

Wipe the entire work area with RNase Zap to destroy RNases. Clean staining dishes, forceps, and slide holders; wrap them with an aluminum foil, and bake them at 350–400°C for approx 4–6 h. Prepare following solutions:

• 1. 4% paraformaldehyde in 1X PBS (DEPC-treated). Store at 4°C.

• 2. 0.1M glycine in 1X PBS (DEPC-treated).

• 3. DEPC-treated deionized distilled autoclaved water (approx 4–5 L).

• 4. Deionized, distilled sterile water (3–4 L).

• 5. Deionized, distilled sterile double-filtered UV-irradiated water (5 mL).

• 6. 10X PBS, 3X PBS, 1X PBS (DEPC-treated).

• 7. Graded alcohols: Dilute 100% alcohol with DEPC-treated water to make 50, 70, and 95% alcohols.

• 8. Proteinase K solution: 1 mg/mL stock solution.

• 9. 100mMTris-HCl.

• 10. Program and clean in situ PCR machine, set oven at 65°C, clean waterbaths, and set the oven’s temperature at 37°C.

• 11. Check the stock of reagents for reverse transcriptase and PCR reaction, RNase-free DNase, Kim wipes, parafilms, plastic coverslips.

• 12. Take 4 (minimum) to 16 (maximum) paraffin-embedded tissue sections mounted on the silanated slides, and place them into the 65°C oven, and melt the paraffin wax overnight.

4.2 Day 1

• 1. Increase the temperature of the oven from 65°C to 80°C to melt the remaining paraffin wax from the slides. Heat the slides at 80°C for 1 h.

• 2. Take two baked staining dishes. Place them in a chemical hood; pour xylene (sufficient enough to cover the slides) into the first dish and put a label on it. Add 100% absolute alcohol into the second dish and put a label on it.

• 3. At the end of the 1–h heat treatment, carefully transfer all slides from the oven, and place them in a slide holder.

• 4. Put slides in xylene for 7–8 min.

• 5. Dry slides in absolute alcohol for 5 min.

• 6. Repeat steps 4 and 5.

• 7. Pour freshly made 4% paraformaldehyde in a clean baked dish and fix the slides for 2–4 h.

• 8. Wash slides twice (5 min each) by gently moving the slide holder up and down in 3X PBS.

• 9. Wash slides twice (5 min each) in 1X PBS.

• 10. Dehydrate slides in graded alcohols: 50, 70, 95, and 100% for 2 min each.

• 11. At this point, slides could be stored at -70°C in a desiccator for at least 6–12 mo, or label the slides with appropriate labels and continue the experiment.

4.2.1 Proteinase K Digestion

• 1. In a clean, RNase-free humidity chamber, add some sterile DEPC-treated water or wet some paper towels and place them on the bottom of the humidity chamber.

• 2. Place the humidity chamber in a 37°C water bath or in an incubator to bring the temperature to equilibrium.

• 3. Rehydrate slides in graded alcohols: 100, 95, 70, and 50% for 2 min each.

• 4. Place a clean sheet of aluminum foil on a bench top work area. Carefully remove each slide from the slide holder. Wipe remaining alcohol around the periphery of the tissue with Kim-wipe and place it on the clean sheet of aluminum foil.

• 5. Place all the slides in a humidity chamber at 37°C for 10 min.

• 6. Thaw the stock solution of proteinase K. Make the following concentrations of proteinase K in different tubes: 5, 10, 15, 20, 25, 30, and 50 μg/mL.

• 7. Optimize conditions for proteinase K digestion by adding 250 μL of different concentrations of the enzyme on different slides of the same tissue block. Incubate for 15 min at 37°C.

• 8. Remove the humidity chamber from the incubator, drain off the enzyme by tilting the slide on a paper towel. Inactivate the enzyme activity by putting the slides into a staining dish with 0.1 M glycine solution for 20 min.

• 9. Wash slides for 5 min in DEPC-treated 1X PBS.

• 10. Dehydrate in graded ethanols: 50, 70, 95, and 100% for 2 min each.

• 11. Observe these slides under the light microscope to check integrity of tissue morphology, fragmentation, breakage, or distortion of the tissue section.

• 12. Make a judgment about the optimum proteinase K concentration for different tissue sections, and select that particular concentration for a future final experiment.

• 13. Sometimes it is a good approach to check the quality of tissue fixation at this stage (see Note 24 ).

• 14. Select the following tissue slides (sectioned from one tissue block) after proteinase K digestion: one with suboptimal digestion; one with optimum digestion; and one with over digestion.

• 15. Add 100 μL of 1X PCR reaction buffer on the top of the each slide, cover the tissue with either a clean plastic or a glass cover slip, and seal with rubber cement (see Notes 18 and 25 ).

• 16. Place these slides in a Thermal Cycler and heat them for 10 min at 95°C.

• 17. Peal off the rubber cement, remove the cover slip, stain the slides for 5 min with 1% Eosin, and observe them under the light microscope. An intact tissue morphology will confirm the quality of slides and fixation (see Note 11 ).

4.2.2 DNase Digestion

• 1. Select slides with optimized proteinase K digestion and follow the steps below for DNase digestion. Prepare in an Eppendorf tube: 1.0 μL RNasin (RNase inhibitor), 11.5 μL UV-irradiated dH₂O, 37.5 U DNase (RQ1 RNase free DNase, Promega Inc., Madison, WI)/(1 U/μL); Total (per slide): 50.0 μL.

• 2. Depending on the size of the tissue sections, add RQ1 RNase free DNase (600– 750 U/mL dH₂O) to the sections (50–100 μL).

• 3. Immediately cover the tissue with a rectangular piece of parafilm (slightly larger than a size of a tissue), with the unexposed side down.

• 4. Incubate the slides overnight (12–16 h) at room temperature (25–28°C) in the humidity chamber.

4.3 Day 2

• 1. Following the incubation, carefully remove the parafilm coverslip from the top of the each tissue section with nuclease-free, sterile forceps.

• 2. Wash the slides twice with DEPC-treated 1X PBS and dehydrate in graded ethanols: 50, 70, 95%, and absolute ethanol, for 2 min each.

4.3.1 Reverse Transcriptase Reaction

• 1. Mark control slides and keep them in Coplin jar with 100 mM Tris-HCl, pH 8.0 until the PCR step.

• 2. Add to each of the other sections 65 μL H₂O + 5 μL oligo (dt). Cover with parafilm and incubate for 10 min at 70°C in the Thermo Cycler.

• 3. Incubate 1 min on ice.

• 4. Prepare RT reaction mix as follows: 250–500 ng Anti-sense primer, 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 0.01 M Dithiothreitol, 20 U (Promega) RNasin, 50 mM, pH 8.3 Tris-HCl, 75 mM KCl, 3 mM MgCl₂, 400 U Superscript II; UV-irradiated dH₂O to total 60 μL.

• 5. Place 60 μL of the RT reaction mix onto each tissue slide.

• 6. Seal tissue sections with clean baked cover slips (20 × 30 mm) using rubber cement (see Note 18 ).

• 7. Leave sealed slides on the bench top for 5–7 min to dry the rubber cement.

• 8. Insert slides into a Thermal Cycler and run a program for RT reaction.

• 9. Incubate slides at 42°C for 1 h immediately followed by raising the temperature to 70°C for 10 min in a Thermal Cycler.

• 10. At the end of the incubation, quickly remove all slides from the Thermal Cycler and place on ice for 1 min.

• 11. Remove cover slips with forceps, wash briefly in DEPC-treated 1X PBS and dehydrate in graded ethanols.

4.3.2 Polymerase Chain Reaction

The conditions for this step have to be set up for every primer by standard tube (in vitro) PCR. The concentration of the different components and the temperature cycle should be the same (or very similar) for both techniques. The number of cycles is lower for in situ PCR, because otherwise one would obtain strong staining that could prevent visualization of the fine morphological details of signal localization.

4.3.2.1 Indirect Method

• 1. Preparation of Taq polymerase-Taqstart antibody complex (see Note 26 ): In 0.5-μL Eppendorf tube, mix the following (for 10 PCR reactions, 50 μL each): 4.4 μL Taqstart-antibody, 17.6 μL Dilution Buffer, 4.0 μL Taq polymerase. Incubate the mixture at 22°C for 5 min.

• 2. Preparation of 10-mM dNTP stock solution: Mix in an Eppendorf: 60.0 μL UV-irradiated dH₂O,10.0 μL dATP (100 mM stock), 10.0 μL dCTP (100 mM stock), 0.0 μL dGTP (100 mM stock), 10.0 μL dTTP (100 mM stock), for a total volume of 100 μL.

• 3. PCR cocktail for indirect in situ PCR: 10.0 μL 10X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl), 3.0 μL MgCl₂ (50 mM stock), 100 pmol Sense strand primer, 100 pmol Antisense primer, 2.5 μL dNTP mix (10 mM stock), 2.0 μL BSA (0.02% stock), 10 U Taq polymerase-Taqstart antibody complex, UV-irradiated filtered dH₂O to make up a total volume of 100 μL.

• 4. Cover the sections with the PCR reaction cocktail (quantity of the reaction mix should be proportional to the size of the tissue).

• 5. Carefully place nuclease-free cover slip (siliconized) on the top of the tissue (see Note 17 ).

• 6. Carefully seal the edge of the cover slip with rubber cement and leave slides on bench top for 5–10 min to dry the rubber cement (see Note 15 ).

• 7. Prepare following control PCR reaction mix for the control slides:

  • a. Omit reverse transcriptase reaction.

  • b. Omit primers in PCR reaction mix.

  • c. Omit Taq polymerase.

  • d. Add only one primer and another unrelated primer.

  • e. Use RNase-treated tissue section for PCR.

  • f. RT positive, PCR positive, and hybridize with an unrelated probe.

  • g. RT negative, PCR negative, and hybridize with a specific sense probe.

• 8. Cover each control slide with an appropriate reaction mix and seal with rubber cement and a coverslip.

• 9. Place all slides into the Thermal Cycler.

• 10. Run the PCR program (see Note 19 ).

• 11. Program the Thermal Cycler to keep the slides at 22–25°C at the end of the PCR cycles (if you plan to leave the slides overnight in the machine).

4.3.2.2 Direct Method

In the case of direct in situ RT-PCR, digoxigenin-labeled 11-dUTP is added in the 4(dNTP) mix. The recommended concentration ratio of digoxigenin 11 -dUTP to dTTP is 1:19 (Boehringer Mannheim).

4.4 Day 3

• 1. Carefully remove the rubber cement and cover slip from each slide.

• 2. Heat the slides at 92°C for 1–2 min either in a Thermal Cycler or on a heat block in order to immobilize the signal.

• 3. Place them in a slide holder and treat with DEPC-treated 1X PBS for 5 min.

• 4. Dehydrate in graded alcohol: 50, 70, 95, and 100% for 2 min each.

• 5. Add 2 ng/μL digoxigenin-labeled oligo probe into the following hybridization solution: 50% deionized formamide, 10 mL 20X SSC, 10X (final concentration) 100X Denhardt solution, 1 mg/mL (final concentration) 10 mg/mL salmon sperm DNA, 1% (final concentration) 10% SDS.

• 6. Add 30–40 μL of probe containing hybridization solution to each tissue section treated with the in situ RT-PCR reaction protocol. Cover with siliconized glass coverslips, seal with rubber cement.

• 7. Heat the slides at 95°C for 10 min in a Thermal Cycler.

• 8. Incubate slides overnight (for 8–12 h) at 37–42°C in a humidity chamber.

4.5 Day 4–Signal Detection

• 1. Remove coverslip and wash each slide in 2X SSC at 48°C for 20 min.

• 2. Soak slides in buffer 1 at room temperature for 10 min.

• 3. Wipe the slide dry around the tissue but keep the tissue area wet. Put onto the each section 200 μL of antidigoxigenin antibody (a Fab fragment), diluted 1:200 in buffer 1. Incubate at least for 2 h at room temperature.

• 4. Wash twice in buffer 1 (5 min each).

• 5. Equilibrate the sections in buffer 2 for 10 min at room temperature.

• 6. Prepare substrate solution: 10.0 mL buffer 2, 27.7 μL Nitro-blue tetrazolium (NBT), 22.5 μL BCIP, 6.0 drops Levamisole (1 mM).

• 7. Carefully wipe the area around each tissue section, place the slides flat in the humidity chamber, and check the horizontal level of the chamber.

• 8. Add 250 μL substrate solution to each tissue section and incubate in the dark. Check under the microscope until the development of the color (bluish purple) is complete. It might take from a few hours to 24–48 h.

• 9. Stop the reaction by immersing in buffer 3 for 5 min.

• 10. Mount the slides in a water soluble mounting medium (e.g., Aqua mount).

• 11. Observe under the light microscope (see Fig. 1 ).

Fig. 1.

Indirect in situ RT-PCR for ras mRNA in normal and neoplastic human breast tissues. (A) and (C) Negative controls for ras message. (B) Detection of ras mRNA signal in normal epithelial cells of mammary duct and lobules. (D) Detection of ras mRNA signal in neoplastic ductal epithelial cells in breast tissue with ductal carcinoma in situ (DCIS).

5 Notes

• 1. To make your own humidity chamber:

  • a. Take a large (8″ × 10″) tightly sealed plastic box.

  • b. Wipe thoroughly with RNase Zap solution to destroy RNases and rinse with DEPC-treated distilled water.

  • c. Cut two or three filter papers or clean paper towels according to the size of the bottom of the plastic box and place flat on the bottom of the box.

  • d. Pour DEPC-treated water uniformly to cover the entire surface.

  • e. Cut several plastic pipets according to the length of the box and place them on the top of the wet filter paper in such a way that they can support approx 20 horizontal glass slides.

  • f. Cover with the lid and wrap the entire box with plastic wrap.

• 2. RNase-free Eppendorf tubes are available commercially. Alternatively, soak regular Eppendorf tubes overnight in DEPC-containing distilled water. Next day, drain the water and place the tubes in a beaker or a glass jar and autoclave. Place the jar in an oven dryer at 80°C for 4–5 h.

• 3. Caution: DEPC is highly carcinogenic! Wear gloves and facial mask before handling. It is safer to purchase it in a solution form (Fluka Chemical Corp., Ronkonkoma, NY) and use a chemical hood while pipeting the aliquot.

• 4. Make a fresh solution for every new procedure, using a chemical hood. Do not use after 15–20 d.

• 5. UV-irradiated, double-distilled water (UV-ddH₂O): Filter autoclaved double-distilled water through 0.22-μm filter. Transfer 1 mL to 1.5-mL capacity RNase-free Eppendorf tubes. Without closing, expose them to UV irradiation for approx 30 min. Close the tubes inside the UV station. Store in -20°C freezer (make approx 20 tubes).

• 6. In the case of direct in situ PCR, digoxigenin-labeled 11-dUTP is added to the above 4(dNTP) mix. The recommended concentration ratio of digoxigenin 11-dUTP to dTTP is 1:19 (Boehringer Mannheim).

• 7. One should check the solubility of signal and counterstain in the mounting medium before selecting it. When water-soluble medium is used (e.g., Aqua mount or Crystal mount), many times, bubbles appear during long storage because of excessive drying. After mounting the slide with aqua mount, seal the edges of the cover slip with a colorless nail polish. This will protect the slide from drying and bubble formation.

• 8. Before starting experiments with human or animal tissue samples, it is extremely important to optimize in vitro experimental conditions. With a purified template nucleic acid, standardize RT and PCR conditions. Check the specificity and crossreactivity of primers and probes. Sometimes it is necessary to alter MgCl₂ concentration under in situ reaction conditions. The blocking reagent for filter hybridization could be different than the in situ protocol. (I use 1 % purified casein solution for filter hybridization and 3% BSA for in situ signal detection.)

• 9. Caution: All specimen handling and subsequent procedures must be carried out using ultra pure, diethylpyrocarbonate-treated deionized distilled autoclaved water (DEPC-water). All solutions, equipment, and glassware used for the pretreatment, RT-PCR protocols, and hybridization stages should be appropriately treated and baked to destroy nucleases, particularly ribonuclease, which is heat-stable.

• 10. If the goal is to detect mRNA, then optimize all positive control reaction conditions with a tissue that is fixed with special care for mRNA protection. This helps in optimizing subsequent reaction conditions and reproducing the data.

• 11. If the tissues come off the slide:

  • a. Examine the original tissue block. It may have dried out. If possible, cut new tissue sections from a better block, or try to use tissue sections derived from the inner part of the tissue block.

  • b. After xylene and alcohol treatment, keep the slides in 4% paraformaldehyde for 4 h or overnight at 4°C in 2% paraformaldehyde.

  • c. Postfix the tissue for 10 min in 4% paraformaldehyde after proteinase K digestion.

  • d. Check on the quality of the silanated slides.

• 12. In formalin-fixed paraffin-embedded tissues, severe nicking of nucleic acid targets may occur during fixation, chemical processing, or from nuclease activity prior to the tissue fixation. Apoptosis could also cause nicks in the DNA. The polymerase action of Taq polymerase enzyme is capable of repairing nicks (1,23). When using “Direct in situ RT-PCR” protocol, detection of false-positive signal is probably a result of the incorporation of labeled nucleotides during nicked DNA repair by Taq polymerase. The appearance of false-positive signal may also be because of a detection system artifact, particularly in those tissues that have high levels of endogenous enzymes, such as alkaline phosphate or peroxidase. Negative results could result from the failure of the in situ RT-PCR reaction or overfixation of the tissue, which could have rendered target nucleic acid inaccessible to the reagents or the probe. Therefore, a large number of controls must be considered for every tissue under investigation (1,23,45).

• 13. A noncrosslinking Streck’s fixative is commercially available from Streck Laboratories (Omaha, NE). This fixative is especially formulated for in situ applications.

• 14. If the primer-negative control slides show intensive nonspecific positive signal, it could be because of the following: When the negative control tissue sections are inadequately digested with proteinase K, all DNA within the tissue does not get completely exposed to the subsequent step of overnight digestion with concentrated RNase-free DNase. Residual DNA that is protected underneath the undigested protein can provide sites for Taq polymerase for DNA synthesis. Repeat the experiment with optimum proteinase K digestion followed by adequate DNase digestion.

• 15. In 1995, Perkin-Elmer introduced a new enzyme rTth-DNA polymerase with a dual activity. It can perform both RT and PCR in the presence of manganese acetate buffer, sense and antisense primers, and nucleotides. This protocol is easier to perform and reduces total in situ RT-PCR reaction time (46).

• 16. When the signal is detected partially on one part of the tissue, or there is no uniformity in the signal amplification pattern, check the following:

  • a. PCR or RT reaction mix might not have covered the entire area of the tissue.

  • b. There might be bubbles underneath the coverslip. When tissues are sealed with a light plastic coverslip, bubbles are formed during the heating steps of PCR cycles. As a result, the reaction mix does not act on or reach uniformly to the entire area of the target tissue. I recommend using thick (minimum 1-mm thickness) siliconized glass coverslips to seal the slides during PCR cycles. If you already have nuclease-free plastic coverslips, seal them with rubber cement, and on the top of the plastic coverslip put a good-size glass coverslip to create some weight in order to prevent bubble formation during PCR cycles.

  • c. Check the level of the humidity chamber. If the antidigoxigenin antibody solution or a substrate solution is not evenly distributed over the tissue, intensity of the signal will differ in the different areas of the tissue.

  • d. A significant delay in fixation after the surgery, and an improper handling of the tissue, can cause partial enzymatic degradation of mRNAs in the tissues.

• 17. Make sure that there are no air bubbles. I usually place 40–50 μL PCR reaction mix on the top of the coverslips. Invert one slide at a time, touch it to the drop of reaction mix, and quickly lift the slide. The coverslip should remain adhered to the slide because of surface tension. Flip the slide, center the coverslip with a clean pipet tip, and gently remove the air bubbles.

• 18. The sealed glass coverslip prevents evaporation of reaction mixture during PCR cycling. For sealing:

  • a. Buy rubber cement in tube form.

  • b. Remove the cap, and insert the mouth of the tube into the small piece of flexible plastic or transparent rubber tube (with same width and approximately 1″ long).

  • c. Insert 1-mL pipet tip and fix it properly inside the flexible tube.

  • d. For sealing slides, place reaction mix onto the top of the coverslips, invert one slide at a time, and touch it to the drop of reaction mixture on the coverslip (the coverslip will remain adhered to the slide), then gently flip the slide.

  • e. Squeeze rubber cement tube very gently around the entire edge of coverslip.

  • f. Let it air-dry completely before placing slide into the PCR machine. Improper sealing can ruin the experiment.

• 19. An example of a PCR cycle would be:

  • a. 96°C. for 2 min.

  • b. 94°C for 1 min.

  • c. 55–60°C for 1 min.

  • d. 72°C for 0.45 s.

  • e. 20–25 cycles.

  • f. 72°C for 10 min.

• 20. Dirty patches or chromogen precipitates on the tissue:

  • a. If, while viewing the slides, you notice deposits of pale yellow crystals or patches over the entire tissue, it may be precipitates of partially dissolved bovine serum albumin from the blocking solution. I prefer to make concentrated blocking solution (10X) and store it in 15-mL tubes. Two hours before the blocking step, thaw two tubes in a 37°C waterbath and vortex several times before adding it to buffer 1.

  • b. Sometimes, the substrate reagents (buffer 2, pH 9.5 with NBT-BCIP) form dark blue-black precipitates during the detection step. Buy the product from a different company. (I have used Enzo Diagnostics, Inc. [Farmingdale, NY], Vector Laboratories, Inc. [Burlingame, CA], and Boehringer Mannheim’s substrates). Filter and prewarm the substrate buffer solution at 37°C after the addition of NBT-BCIP.

• 21. If there is too much signal on the entire slide, check the following:

  • a. Concentration of the probe (it should be 2 ng/μL). Under standardized posthybridization wash conditions, a very highly concentrated unbound probe will not be washed away. Recalculate the probe concentration and increase the stringency of wash conditions.

  • b. Do not exceed the incubation time (>2 h) with diluted (1:200) antidigoxigenin antibody solution.

  • c. Destroy endogenous alkaline phosphatase by treating the slides with either 0.02 N HCl at room temperature for 8 min or 20% acetic acid for 20–30 s at 4°C for 20 s.

• 22. When control slides show optimum signal, but there is poor or no signal in the experimental slides, this may be because of:

  • a. Degradation of the target nucleic acid sequences during initial fixation.

  • b. Overfixation with 4% paraformaldehyde. Check the concentration of paraformaldehyde, reduce the fixation time, andreoptimize the proteinase K digestion.

  • c. Poor sealing of slides causing the evaporation of RT and PCR reagent mix from the slide during reaction cycles.

  • d. Improper preparation of reaction mixtures or suboptimal concentration of MgCl₂.

  • e. Inactive or old batch of enzymes.

  • f. Extremely stringent wash conditions. (Oligoprobes can be washed away under very high stringency.)

  • g. Drying of the antidigoxigenin antibody. (Always check the surface level of the humidity chamber before the addition of antidigoxigenin antibody solution.)

  • h. Selection of a wrong batch of tissues.

• 23. To remove coverslips from the slides:

  • a. If permount is used as a mounting medium, a slide should be soaked in xylene for 10–15 min. The treatment with xylene dissolves permount and the coverslip comes off.

  • b. When slides are permanently mounted with Aquamount, it is necessary to soak slides in hot water (85°C) for 30–40 min to remove the coverslip.

• 24. It is important to proceed further only if tissue retains its morphology intact after proteinase K digestion. Discontinue the experiment if the tissue is distorted. This saves your valuable time and reagents.

• 25. The size of coverslip should be bigger than the tissue and its width should be less than the width of the glass slide. I prefer 20 × 30-mm or 20 × 40-mm glass coverslips. Proper airtight sealing prevents evaporation of reagents and gives uniform signal over the entire surface of the tissue.

• 26. Preparation of Taq polymerase-Taq-start antibody complex: I mix 1:1 Taq-start antibody: Taq polymerase enzyme and incubate at 22°C for 10 min.