Introduction

In situ hybridization is a well-established method used to detect messenger RNA in tissue and is used widely. In contrast, reverse transcription (RT) in situ polymerase chain reaction (PCR [1,2]) has remained in the developmental stage, and the technique requires further refining before it can be used for specific applications. Although many laboratories described applications of RT in situ PCR in various fields during the 1990s, there also has been considerable criticism regarding the method.

As shown in Fig. 1, there are several variations of RT in situ PCR In so-called "direct detection," primers, which are used for PCR amplification,

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.

Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

Fig. 1. Schematic representation of principal steps for direct or indirect RT in situ PCR. In both the direct and indirect methods, mRNA is first converted to cDNA (1, RT step). Subsequently, PCR amplification is performed by using the cDNA as a templates (2, PCR amplification step). In the direct method, amplified products harboring with labeled nucleotide or primers are detected after immunohistochemical staining (3, immunohistochemical detection step). By contrast, in the indirect method, amplified PCR products are hybridized with specific labeled primers (3, hybridization step), then detected by immunohistochemical staining (step 4).

Fig. 1. Schematic representation of principal steps for direct or indirect RT in situ PCR. In both the direct and indirect methods, mRNA is first converted to cDNA (1, RT step). Subsequently, PCR amplification is performed by using the cDNA as a templates (2, PCR amplification step). In the direct method, amplified products harboring with labeled nucleotide or primers are detected after immunohistochemical staining (3, immunohistochemical detection step). By contrast, in the indirect method, amplified PCR products are hybridized with specific labeled primers (3, hybridization step), then detected by immunohistochemical staining (step 4).

are labeled with biotin, digoxigenin (DIG), or other fluorescein reagents. Alternatively, the labeled nucleotide, DIG-11-UTP (dTUP coupled with, DIG via an alkali-labile ester-bond) is incorporated into amplified products during PCR. In these direct methods, the labeled products can be visualized at the end of the reaction. In "indirect detection" (3), PCR is performed with unlabeled primers and dNTP in a similar manner to standard RT-PCR conducted in a tube and, subsequently, amplified products are hybridized with a labeled probe. Then, specifically hybridized probes are observed in a manner similar to that used in Southern or Northern hybridization.

Direct RT in situ PCR

Direct RT in situ PCR

Indirect RT in situ PCR

Indirect RT in situ PCR

a. Correct amplification products b. Non-specific amplication products

Fig. 2. Nonspecific amplification which may cause "false-positive" in direct in situ PCR method. It is possible to detect the nonspecific PCR products (indicated as b) by the direct RT in situ PCR, whereas the hybridization step could neglect the nonspecific products in indirect RT in situ PCR. Therefore, indirect RT in situ PCR could specifically detect the correct amplification products (a).

a. Correct amplification products b. Non-specific amplication products

Fig. 2. Nonspecific amplification which may cause "false-positive" in direct in situ PCR method. It is possible to detect the nonspecific PCR products (indicated as b) by the direct RT in situ PCR, whereas the hybridization step could neglect the nonspecific products in indirect RT in situ PCR. Therefore, indirect RT in situ PCR could specifically detect the correct amplification products (a).

The direct method is shorter, less expensive, and minimizes the manipulation of the tissues if proper signals are obtained (4). However, compared with direct methods, there is an advantage in indirect RT in situ PCR with regard to specificity (3,5). As demonstrated in Fig. 2, we cannot discriminate the nonspecific signals from correct signals in direct RT in situ PCR It is not uncommon to observe nonspecific bands, which are of an undesired length, in standard RT-PCR. It also is not uncommon to observe several nonspecific bands or even smears in inappropriate PCR conditions. These phenomena can occur as the result of poor specificity of the primer sets used or excess quantities of Taq polymerase or MgCl2. As described herein large quantities of Taq polymerase and MgCl2 usually are used in RT in situ PCR, compared with standard RT-PCR, to increase the sensitivity. Therefore, we suggest that indirect RT in situ PCR more reliably excludes nonspecific amplification by using hybridization steps after PCR.

The application of indirect RT in situ PCR consists of three major steps. First, complementary (c)DNA is synthesized in situ from mRNA by reverse transcriptase. Second, synthesized cDNA is amplified with primers by in situ

PCR Third, amplified products are visualized by hybridization with labeled probes.

The three major problems in RT in situ PCR are (1) mRNA is easily degenerated by RNase, which can contaminate samples and originate from almost anywhere; (2) it is necessary to take controls to verify the results; and (3) in general, the sensitivity of RT in situ PCR is not as high as expected because of poor amplification of gene products, which is estimated to be just 100-fold in tissue sections.

With respect to the first problem, it is important to consider the degree degradation of mRNA before initiating the RT process. In the detection of murine mRNA by in situ hybridization, the tissues usually are fixed using circulation or immediately frozen followed by fixation. This careful handling of specimens may prevent the degradation of mRNA, which occurs during the delay between tissue isolation and fixation. In human tissues, the delay between tissue isolation and fixation typically is long and, thus, it often is difficult to obtain sufficient signal intensity in most routinely processed pathological tissues sections by in situ hybridization. Although the sensitivity of RT in situ PCR can be greater than simple in situ hybridization, we believe that inappropriately processed paraffin-embedded tissue sections are not suitable to detect mRNA in situ.

In the second problem, we have to discriminate true signals from fake signals, which are arising from hybridization with genomic DNA. We found this especially problematic when trying to detect viral RNA by RT in situ PCR However, we believe the same problem is less likely to occur in detecting chimeric genes by RT in situ PCR This is because the length of genomic DNA that represents chimeric mRNA is very long and is difficult to amplify by RT in situ PCR It generally is accepted that the limited length, which we detect by using paraffin-embedded tissue as a template, may be less than 500 to 600 base pairs in standard PCR (6). Furthermore, most tumors may also contain non-neoplasmic cells, including infiltrating leukocytes, which also may provide quality control for RT in situ PCR

In the third problem, the mechanism, which is responsible for the poor efficiency of RT in situ PCR, still remains obscure. However, the so-called "diffusion artifact" may be a major cause of this poor efficiency. During the PCR steps, the amplification products may not remain in the locality of the initial template. A significant proportion of amplified products may float into the reaction buffer and be used as template by subsequent PCR steps. Overlaying tissue sections with a thin layer of agarose may help to decrease the effects of the "diffusion artifact" (7); however, it may reduce the permeability required to allow polymerase to reach the template. We speculate that the reliability of RT in situ PCR would be greatly increased if the effects of the diffusion artifact could be minimized.

Fig. 3. Schematic representation of primers sets used in the current protocol.

Here, we describe our current protocol to detect a chimeric oncogene in human soft tumor, synovial sarcoma, by RT in situ PCR Synovial sarcomas are typified by a unique chromosomal translocation t(X;18)(p11.2;q11.2) that results in fusion of the SYT gene on chromosome 18 with the SSX1 or SSX2 gene in Xp11.2 and, consequently, production of the chimeric, SYT-SSX proteins (Fig. 3 [8,9]). It is suggested that SYT-SSX is involved directly in tumorigenicity of synovial sarcoma by interfering with the function of chromatin remodeling (10,11). Synovial sarcoma is not restricted to the synovium and can actually occur at any site. Unfortunately, histological diagnosis of synovial sarcoma, especially that of a monophasic fibrous type and/or in an extrasynovial site, can sometimes be difficult. As a result, it is desirable to establish dependable methods to reliably identify synovial sarcoma using pathologically processed specimens. Previously, we tried to detect chimeric, SYT-SSX mRNA by simple in situ hybridization; however, sufficient signals were not detected in almost all paraffin embedded specimens, which may have been attributable to insufficient quantities of SYT-SSX mRNA in synovial sarcoma cells.

2. Materials

2.1. Preparation of Tissue Sections (see Note 1)

1. MAS-coated precleaned Micro slide glass (Matsunami, Japan; see Note 2).

2. 10% Formalin neutral buffer solution (Wako Co. Ltd, Japan).

3. Histoprep 580, paraffin (Wako Co. Ltd).

2.2. Reverse Transcriptase

1. Proteinase K (RNase-free grade, Takara, Japan).

2. RNase-free DNase: 5 U RNase-free, DNase in 20 mMTris-HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 0.1 mM CaCl2.

3. RNA long and accurate (LA) PCR kit (AMV) Ver. 1.1 (Takara; see Note 3).

4. Primers, sense: 5'-CAACAGCAAGATGCATACCA-3' (forward primer for SYT); antisense: 5'-CACTTGCTATGCACCTGATG-3' (reverse primer for SSX1).

5. RT reaction buffer: 10 mMTris-HCl, 50mMKCl, 1.5 mMMgCl2, 25mMdNTPs.

6. 100 nM SYT-SSX primer sets, 50 U RNase inhibitor, and 400 U Moloney murine leukemia virus reverse transcriptase.

7. Hybaid OmniSlide In-Situ Thermal Cycler (Hybaid Ltd, UK; see Note 4).

8. Hybaid EasiSeal (Hybaid Ltd).

2.3. Polymerase Chain Reaction

1. AmpliTaq, GOLD polymerase (Applied Biosystems, CA).

2. 10X PCR buffer: 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 0.01% (w/v) gelatin, dNTPs, and 25mM MgCl 2 solution are attached to this enzyme.

3. Hybaid OmniSlide In-Situ Thermal Cycler (Hybaid Ltd).

4. Hybaid EasySeal (Hybaid Ltd).

5. Primers. sense: 5'-GCTACGGTCCTTCACAGGGTGGTCCAGG-3' (nested primer for SYT); antisense: 5'-AGATGCTTCTGACACTCCCTTCGAATC-3' (nested primer for SSX1).

6. Washing solution: 10 mM Tris-HCl, pH 7.5.

2.4. Labeling and Hybridization of Probe

2. Trizol Reagent (GibcoBRL, Rockville, MD).

3. DIG high prime-labeling and detection kit (Boehringer Mannheim GmbH, Germany).

4. DIG Easy Hyb (Boehringer Mannheim GmbH).

5. 20X standard saline citrate (SSC): 3 MNaCl, 0.3 Msodium citrate (pH 7.4).

6. 2X SSC: Dilute 20X SSC 10-fold with distilled water.

7. 0.5X SSC: Dilute 20X SSC 40-fold with distilled water.

2.5. Immunohistchemical Detection of DIG

1. DIG high prime-labeling and detection kit (Boehringer Mannheim GmbH; see Note 5).

2. Maleic acid buffer: 0.1 Mmaleic acid, 0.15 MNaCl. Adjust with NaOH to pH 7.5.

3. Detection buffer: 0.1 MTris-HCl, 0.1 MNaCl, 50 mMMgCl2, pH 9.5.

3. Methods

A novel cell line, designated HS-SYII, was derived from a synovial sarcoma of the monophastic fibrous type and harbors SYT-SSX1 transcripts (12). We used cultured H S-SYII cell clotting to establish the current protocol. Briefly, 1 X 107 cells are collected by centrifugation, fixed in formalin for 16 h at room temperature, and finally embedded in paraffin as surgically resected tissues. Prepared tissue sections were used as positive controls, whereas surgically resected other soft part tumor tissues were used as negative controls. We bypassed the RT step to avoid the possibility of DIG-labeled probe hybridizing with SYT-SSX1 mRNA in situ. In our experimental conditions, we were unable to detect any significant signals without RT steps even in, H. S.-SYII cell clots. We believe that the amount of HS-SYII mRNA in paraffin-embedded synovial sarcoma tissues is insufficient for detection by simple in situ hybridization, or alternatively is degraded after the RT steps of the current protocol.

3.1. Preparation of Tissue Sections

1. Immediately after resection, the tissues are cut and fixed in 10% buffered formalin for 10 to 16 h at room temperature (see Note 6).

2. Tissues are cut into 6- to 8-pm-thick sections (see Note 7). The sections are deparaffinized with lemosol and dehydrated with ethanol. For frozen tissues, the tissues are cut immediately, and then fixed in ethanol at room temperature for 5 min.

3. The sections are digested with 40 pg/mL proteinase K in, phosphate-buffered solution for 20 min at room temperature (see Note 8). Proteinase K is then inactivated by heating at 95°C for 5 min.

3.2. RT In Situ Step

1. Tissue sections are subsequently treated with RNase-free DNase at 37°C for 20 min (see Note 9). Inactivate DNase by incubating the specimens at 75°C for 10 min.

2. The RT procedure is conducted using reaction buffer. Fifty microliters of reaction buffer is added to tissue sections, sealed with Hybaid Easyseal, and incubated at 37°C for 10 min at 42°C for 30 min, 95°C for 5 min, and chilled on ice. The tissues are fixed with 80% ethanol at room temperature for 2 min (see Note 10).

3.3. PCR In Situ Step

1. After washing in washing solution, 50 pL of reaction mixture containing 5 U of AmpliTaq Gold polymerase (Perkin Elmer; see Note 11), 200 pM of each deoxynucleotide triphosphate, and 50 nM of SYT-SSX primer (nested primers) is added to the glass slide and subsequently covered with an EasySeal cover. The final concentration of MgCl2 is 4.5 pM (see Note 12).

2. The PCR step is performed by heating at 95°C for 7 min, followed by 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Final elongation of PCR product is performed at 72°C for 20 min.

3. Tissues are incubated in PBS for 5 min and subsequently fixed in 100% ethanol for 5 min at room temperature (see Note 13).

3.4. Labeling of cDNA Probes and Hybridization Step

1. DIG easy hybridization buffer is added and the tissues are incubated at 55°C for 1 h.

2. SSX-SYT chimeric 200-bp cDNA was obtained from cultured HS-SYII with nested primers by using RNAzol and the RNA LA PCR kit.

3. Amplified SSX-SYT cDNA is labeled with DIG according to the random primed labeling procedure by using DIG high prime-labeling kits. Briefly, 1 pg of S SX-SYT cDNA in 16 pL of sterile water is heated to 95°C for 10 min and quickly chilled on ice. Four microliters of DIG high prime mixture is added to SSX-SYT cDNA and centrifuged briefly. After 16 h of incubation at 37°C, the mixture is heated at 95°C for 10 min and quickly chilled on ice.

4. The DIG-labeled cDNA is mixed with DIG easy hybridization buffer and then heated to 55°C.

5. Diluted DIG-labeled cDNA is added to tissues (see Note 14). The tissues are incubated at 55°C overnight (14-16 h).

3.5. Posthybridization Washes

1. Tissue slides are washed twice for 5 min with a solution containing 2X SSC and 0.1% SDS at room temperature.

2. The tissue slides are then washed twice for 15 min with a solution containing 0.1X SSC and 0.1% SDS at 55°C.

3.6. Immunohistochemical Detection of Labeled Probe

1. The tissue slides are washed with maleic acid buffer for 5 min at room temperature.

2. The tissues are incubated with blocking buffer for 30 min at room temperature.

3. The tissues are incubated with diluted anti-DIG-AP conjugate for 1 h at room temperature (see Note 15).

4. The tissue section is washed twice for 15 min with maleic acid buffer.

5. The tissue section is washed for 5 min with detection buffer.

6. The tissues are incubated with freshly prepared color solution at room tempera-hhhture (see Note 16).

7. Finally, the tissues are washed with phosphate-buffered saline. Representative examples are shown in Fig. 4.

4. Notes

1. Fixation of the tissues is the most important process for RT in situ PCR It is ideal to determine the fixing condition by using cultured cell clots in which the desired mRNA has been preliminarily identified.

2. Any glass slide can be used as an alternative to that described; however, glass slides in which tissues were not detached during any of the steps of the procedure and possess little or no contamination of RNase should be preferentially selected.

3. RT, buffer, dNTPs, and RNase inhibitors are included in this kit.

4. Martinez et al. (4) reviewed a few models of thermocyclers for in situ PCR and selected this OmniSlide system based on ease and flexibility of operation.

5. This kit contains specific antibody to DIG 10X blocking solution, and color-substrate solution.

6. We sometimes encountered difficulties in detecting signals by RT in situ PCR when fixing large tissues (more than 1 cm3) without adequate precutting. These difficulties may be caused by degradation of mRNA before fixation. Most of the

Fig. 4. Representative results of RT in situ PCR. A synovial sarcoma, in which SYT-SSX chimeric gene was detected by standard RT-PCR in tube, was stained according to the current protocol. Spindle tumor cells (A, hematoxylin and eoxin staining, original magnification, x400) were stained by RT in situ PCR (B, x200; C, x400). Note the positive signals in cytoplasm (C, arrow). As a negative control, the RT step was bypassed (D, x400). Omission of the primers in the PCR mixtures resulted in elimination of the signals (E, x400). In D and E, nuclei were stained with hematoxilin after RT in situ PCR to identify the cells. Cell clot of HS-SY II also was stained with current protocol (G, x400, white arrows indicate cytoplasmic staining). Omission of the primers again eliminated the signals (H, x400, hematoxylin staining was added). Spindle cell tumor of Schwannoma (I, hematoxylin and eosin staining, x400) was not stained by, RT in situ PCR (J).

Fig. 4. Representative results of RT in situ PCR. A synovial sarcoma, in which SYT-SSX chimeric gene was detected by standard RT-PCR in tube, was stained according to the current protocol. Spindle tumor cells (A, hematoxylin and eoxin staining, original magnification, x400) were stained by RT in situ PCR (B, x200; C, x400). Note the positive signals in cytoplasm (C, arrow). As a negative control, the RT step was bypassed (D, x400). Omission of the primers in the PCR mixtures resulted in elimination of the signals (E, x400). In D and E, nuclei were stained with hematoxilin after RT in situ PCR to identify the cells. Cell clot of HS-SY II also was stained with current protocol (G, x400, white arrows indicate cytoplasmic staining). Omission of the primers again eliminated the signals (H, x400, hematoxylin staining was added). Spindle cell tumor of Schwannoma (I, hematoxylin and eosin staining, x400) was not stained by, RT in situ PCR (J).

suitable fixed samples in our experiments were those where the tissues were immediately frozen in liquid nitrogen and cut into 6- to 8-|im thick sections and subsequently fixed in ethanol for 5 min at room temperature. Alternatively, tissues may be fixed in 10% buffered formalin or 1-4% paraformaldehyde for 12 to 16 h or 20 min, respectively, at room temperature.

7. Special care should be taken to avoid RNase contamination during tissue preparation. This applies to all reagents and equipment, such as cutting knives, which should be pretreated to inhibit RNase activity or acquired new from the manufacturer.

8. In order to gain sufficient signal intensity, the tissues are cut into 6- to 8-|im thick sections. The permeabilization step facilitates the entry of reagents into the tis sues. There is no standard permeabilization method, including the selection of proteinase. However, we use proteinase K because we expect that it also directed to RNase in the tissue section.

9. We may omit this step during preliminary experiments in cases where we do not need to discriminate between the localization of RT in situ PCR signals. When sufficient signal intensity is detected in the cytoplasm, this step may be added to remove any signal because of amplification arising from genomic DNA.

10. In many protocols, extensive washing with various concentrations of SSC is performed after the RT reaction. We did not find any benefit in this wash step because diffusion of cDNA out of the tissue could result in poor PCR efficiency. Instead of extensive washing, we found that incubation of the tissue section in 80% ethanol was able to fix the cDNA in situ and wash the buffer out of the tissues.

11. We used hot-start Taq polymerase to reduce nonspecific binding of primers to the cDNA template.

12. It generally is accepted that the efficiency of in situ PCR is less than standard PCR performed in a tube. To achieve sufficient amplification, it is important to increase the final concentrations of Taq polymerase, MgCl2, and/or primers.

13. This postamplification treatment also may remove a part of products on the outside of the cells. Amplified products formed during PCR may readily diffuse from the site in which the original cDNA template was located. We speculate that the poor efficiency of RT in situ PCR may be caused by this phenomenon, also known as the diffusion artifact Once the products have floated into the reaction buffer, it can be a template for subsequent PCR amplification, thereby seriously decreasing the intensity of the final signal at the original site. Overlaying tissue sections with a thin layer of agarose may help to decrease the diffusion artifact, although we were unable to detect any significant benefit in our experiments.

14. Dilution rates are dependent on individual experimental conditions. However, we usually diluted labeled cDNA probe 20-fold with DIG easy hybridization buffer.

15. We usually dilute the antibody 1000-fold. However, the dilution rate should be optimized for individual experimental conditions.

16. During color development, do not shake the tissues. Exposure to light should be restricted as much as possible. The color development is usually performed by overnight incubation but can be shortened by 2 to 3 h when sufficient signal intensity is observed.

References

1. Nuovo, G. J. (1996) PCR In situ Hybridization Protocols and Applications (2nd ed). Philadelphia, New York, Lippincott-Raven.

2. Nuovo, G. J. (1997) PCR In situ Hybridization Protocols and Applications (3rd ed). Philadelphia, New York, Lippincott-Raven.

3. Long, A. A., Komminoth, P., Lee, E., and Wolfe, H. F. (1993) Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Histochemistry 99, 151-162.

4. Martinez, A., Miller, M. J., Quinn, K., Unsworth, E. J., Ebina, M., and Cuttitta, F. (1993) Non-radioactive localization of nucleic acids by direct in situ PCR and in situ RT-PCR in paraffin-embedded sections. J. Histochem. Cytochem. 43, 739-747.

5. Komminoth, P. and Long, A. A. (1993) In-situ polymerase chain reaction: an overview of methods. Applications and limitations of a new molecular technique. Virchow Arch. (B) 64, 67-73

6. Wickham, C. L., Boyce, M., Joyner, M. V., et al. (2000) Amplification of PCR products in excess of 600 base pairs using DNA extracted from decalcified, paraffin wax embedded bone marrow trephine biopsies. Mol. Pathol. 53, 19-23.

7. Chiu, K. P., Cohen, S. H., Morris, D. W., and Jordan, G. W. (1992) Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J. Histochem. Cytochem. 40, 333-341.

8. Clark, J., Rocques, P. J., Crew, A. J., et al. (1994) Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat. Genet. 7, 502-508.

9. Crew, A. J., Clark, J., Fisher, C., et al. (1995) Fusion of STT to two genes, SSX1 and SSX2, encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma. EMBO J. 14, 2333-2340.

10. Nagai, M., Tanaka, S., Tsuda, M., et al. (2001) Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2 alpha. Proc. Natl. Acad. Sci. USA. 98, 3843-3848.

11. Kato, H., Tjernberg, A., Zhang, W., et al. (2002) SYT associates with human SNF/SWI complexes and the C-terminal region of its fusion partner SSX1 targets histones. J. Biol. Chem. 277, 5498-5505.

12. Sonobe, H., Manabe, Y., Furihata, M., et al. (1992) Establishment and characterization of a new human synovial sarcoma cell line, HS-SY-II. Lab. Invest. 67, 498-505.

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