Journal of Histochemistry and Cytochemistry, Vol. 45, 1659-1664, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Detection of Phosphatidylinositol Glycan Class A Gene Transcripts by RT In Situ PCR Hybridization: A Comparative Study Using Fluorescein, Texas Red, and Digoxigenin-11 dUTP for Color Detection

Susan Simona, Birgit Reiperta, Martha M. Eibla, and Alexander Steinkasserera
a Immuno A.G., Department of Oncogenic Viruses, Vienna, Austria

Correspondence to: Susan Simon, Immuno A.G., Dept. of Oncogenic Viruses, Benatzkygasse 2-6, A-1220 Vienna, Austria.


  Summary
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Gene-specific probes labeled with fluorescein, Texas Red, and digoxigenin-11 dUTP (DIG) were used for RT in situ PCR hybridization to detect PIG-A gene (phosphatidylinositol glycan class A) transcripts. The PIG-A gene is responsible for biosynthesis of the glycosylphosphatidyl-inositol (GPI) anchor. Lack of GPI anchor expression due to mutations can cause an acquired clonal hematologic disorder called paroxysmal nocturnal hemoglobinuria (PNH). In this RT in situ PCR study, two types of labeling methods, a direct method (using fluorescein and Texas Red) and an indirect method (using DIG-11 dUTP) were compared. Both were successfully applied to detect and localize the PIG-A gene transcripts within single cells of the cell lines AA2, H9, and JY. Furthermore, similar results for sensitivity and reproducibility were obtained. Advantages and disadvantages of the different labeling techniques are discussed. In addition, peripheral blood mononuclear cells from PNH patients were also included in this study. (J Histochem Cytochem 45:1659-1664, 1997)

Key Words: RT in situ hybridization, PIG-A gene, paroxysmal nocturnal, hemoglobinuria


  Introduction
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Summary
Introduction
Materials and Methods
Results and Discussion
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Literature Cited

In recent years, nonradioactive in situ hybridization techniques have frequently been used to map specific genes on chromosomes and to detect viral and bacterial genomes in infected tissues. Because of the inherent technical difficulties associated with this method, the examination of mRNA expression using RNA in situ hybridization has gained less attention. However, reverse transcriptase (RT) in situ PCR has greatly improved this method for the detection of RNA transcripts within intact cells (Nuovo 1993 , Nuovo 1997 ).

In this study we used the RT in situ PCR hybridization technique as a tool to study the expression of phosphatidylinositol glycan class A (PIG-A) in single cells. The PIG-A gene transcripts are responsible for biosynthesis of the glycosylphosphatidyl-inositol (GPI) anchor. More than 20 cell surface proteins are known to be linked to the cell membrane through such an anchor (Rosse 1990 ; Rotoli et al. 1993 ). Among them are two complement regulatory proteins, the decay accelerating factor (DAF) CD55 and the membrane inhibitor of reactive lysis (MIRL) CD59 (Miyata et al. 1994 ; Yamada et al. 1995 ).

Lack of GPI anchor expression can cause an acquired clonal hematological disorder called paroxysmal nocturnal hemoglobinuria (PNH) (Takeda et al. 1993 ; Miyata et al. 1994 ; Yamada et al. 1995 ). Abnormalities of the PIG-A gene have been demonstrated in PNH patients in whom erythrocytes, polymorphonuclear cells, lymphocytes, and platelets are deficient in GPI anchor proteins (Rosse 1990 ; Rotoli et al. 1993 ).

GPI-linked molecules can be assayed by flow cytometry (FACS) and in cellular extracts by ELISA or RIA techniques (Ninomiya et al. 1982 ). Moreover, cellular localization can be determined by immunological or histological methods. However, study of gene expression at the transcriptional level by RT in situ PCR hybridization constitutes one of the most powerful techniques.

In this study PIG-A gene expression was analyzed in three different lymphocytic cell lines (AA2, H9, and JY) using two types of labeling methods, a direct method and an indirect method. In addition, peripheral blood mononuclear cells derived from PNH patients were analyzed with a direct labeling technique.

In the direct labeling procedure, two different fluorochromes, fluorescein and Texas Red, were coupled to the oligonucleotide probes. Probe and target hybrids were visualized by fluorescence microscopy. In the indirect procedure, the reporter molecule digoxigenin (DIG) 11-dUTP was introduced enzymatically into the probes and was detected by anti-digoxigenin (anti-DIG) antibodies conjugated to alkaline phosphatase. The labeled amplicon was detected with brightfield microscopy.

Similar results regarding sensitivity and reproducibility were obtained for each labeling and detection method used. The advantages and problems associated with the different labeling techniques will be discussed. Finally, our results provide support for the application of RT in situ PCR in characterizing the role of PIG-A gene expression in PNH and other GPI-related disorders.


  Materials and Methods
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Materials and Methods
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Cell Culture and Cell Preparation: Cell Lines
The cell lines AA2 and H9 were obtained from the NIH AIDS Research and Reference Reagent Program (catalogue Nos. 135 and 87, respectively). JY cells were kindly provided by Dr. Timothy Springer (Center for Blood Research Institute, Harvard Medical School, Boston). All cells were grown in Ultra CULTURE serum-free medium (BioWhittaker; Walkersville, MD) supplemented with antibiotics (penicillin 1000 IU/ml and streptomycin 10,000 µg/ml; GIBCO, Vienna, Austria) and 1 mM sodium pyruvate. Cells were cultured in T-75 flasks (Nunc; Naperville, IL) at 37C in a humidified atmosphere containing 95% air/5% CO2. The medium was changed at 3-day intervals. The cells were fixed at room temperature (RT) for 20 min in 4% (w/v) paraformaldehyde (pH 7.4) in a total volume of 5 ml. The fixed cells were washed twice with PBS at RT and their density was adjusted to 1 x 105 cells/ml. Two hundred µl of cells was then transferred onto poly-L-lysine-coated chamber slides (Nunc) and allowed to adhere at room temperature for 2 hr as described (Wilkinson 1992 ). Nonadherent cells were washed away with PBS, and the slides were then air-dried and stored in a dust-free environment until use.

Cells from PNH Patients
Heparinized blood samples from PNH patients were obtained from the Red Cross (Department of Haematology, Budapest, Hungary) and were separated by Ficoll-Hypaque gradient centrifugation (Boyum 1974 ). Peripheral blood mononuclear cells (PBMCs) were washed three times in PBS and 1 x 105/ml were plated onto poly-L-lysine-coated chamber slides and allowed to adhere as described above.

Treatment of the Cells
Cells were sequentially treated with 0.1% Triton X-100/PBS (pH 7.4) and 0.2 M HCl, 10 min each, and then with 1 µg/ml proteinase K (Boehringer; Mannheim, Germany) in buffer (10 mM Tris, pH 8.0, 2 mM CaCl2, 50 mM EDTA) at 37C for 6 min to permeabilize cell membranes. This reaction was stopped by rinsing the slides with ice-cold 0.1 M glycine/PBS (pH 7.2) for 10 min. DNase treatment was then performed overnight in a humidified chamber at 37C in a total volume of 10 µl using 10 U DNase (RNase-free; Boehringer) in buffer (105 mM sodium acetate, 5 mM MgSO4). Standard precautions were used to prevent RNase contamination.

Probe Preparation and Primers Used for Analysis of the PIG-A Gene
The custom-made synthetic oligonucleotides (GENSET; Paris, France) used in this study were labeled with fluorescein or Texas Red (Table 1). Synthetic oligonucleotide probes have the advantage over riboprobes in that they are more stable and also smaller, which facilitates penetration into the cells. Primers A1 and B7 were labeled with fluorescein, primers A5 and B12 were labeled with Texas Red [for primer orientation within the PIG-A gene transcript see Figure 1; for sequence of all primers used in this study (according to Yamada et al. 1995 ) see Table 1]. The probe-target hybrids, generated by direct labeled RT in situ PCR, were immediately visualized by fluorescence microscopy.



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Figure 1. Schematic representation of the PIG-A cDNA and the primers used for RT in situ PCR hybridization (not to scale). The specific primers are indicated above the arrows. Numbers in boxes represent exons 2-6. UTR, untranslated region.


 
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Table 1. PIG-A gene analysis

In indirect labeling experiments in which the unlabeled primer set A1-B3 was used, DIG was included in the PCR amplification solution. The labeled amplicons were detected by immunohistochemistry using high-affinity anti-DIG antibodies (1:50 dilution) conjugated to alkaline phosphatase (Boehringer; Vienna, Austria). The alkaline phosphatase conjugates were then visualized with the alkaline phosphatase substrates nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indolyl-phosphatase (BCIP). To eliminate nonspecific backround signals, the blocking solution No. 1096176 (Boehringer) was used as recommended by the manufacturer.

RT In Situ PCR Hybridization
The RT in situ PCR hybridization method for the identification of mRNA within single cells using an indirect labeling technique has been previously described by Nuovo 1993 . Our method is an adaptation of this protocol, using DIG-11 dUTP. In addition, we have used a direct labeling technique, with fluorescein and Texas Red as two different staining reagents.

In Situ Reverse Transcription
The slides were placed on the flat heating block of a thermal cycler (Biomed Thermocycler 60; Biotrade, Vienna, Austria) and held at 42C for 1 hr. To the cells was added 20 µl of RT buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2) containing 10 mM dithiothreitol, 200 U cloned Moloney murine leukemia virus reverse transcriptase (RT) (GIBCO), 40 U RNase inhibitor (Boehringer), and 1 mM of each dNTP. Primer B13 (50 pmol/ml) was used for reverse transcription. To prevent dessication of the cells, Self-Seal reagent (MJ Research; Watertown, MA) was added to the PCR mixture and the slides mounted with plastic coverslips. The RT reaction was then stopped by heating the block for 5 minutes at 94C.

Amplification of the PIG-A Gene
Slides were preheated (70C) using the thermal cycler (Biomed Thermocycler 60; Biotrade, Vienna, Austria). The PCR amplification solution, containing the different primer sets A1-B7 (fluorescein), A5-B12 (Texas Red), and A1-B3 (digoxigenin 11-dUTP) were kept at 95C for 15 min to eliminate primer oligomerization and to activate the AmpliTaq Gold enzyme (Perkin Elmer; Vienna, Austria), which is in an inactive form at room temperature. Twenty µl of the PCR amplification solution containing 2.5 µl of 10 x PCR buffer (final concentrations 2.25 mM MgCl2, 250 µM dNTPs), 5 U of AmpliTaq Gold polymerase, and 10 pmol/ml of each primer were laid on top of the cells. In the amplification solution, the diethylpyrocarbonate (DEPC; Sigma, Vienna, Austria) H2O was substituted by Self-Seal reagent. After an initial denaturation step at 94C for 3 min, 20 cycles were performed using the following cycle steps: denaturing (94C); annealing (55C); extension (72C), each for 1 min. In negative control reactions, primers were omitted.

Posthybridization Treatment
Nonspecific hybridization of primers in the RT in situ PCR reaction can give rise to high background. To reduce this, we included a high-stringency wash using 1 x SSC (0.15 mM NaCl, 0.015 mM sodium citrate) containing 0.1% BSA, (Sigma No. 4628) at 54C for 10 min as described previously (Nuovo 1993 , Nuovo 1997 ). In experiments using fluorescein and Texas Red, signals were detected using a Nikon fluorescence microscope at 500-fold magnification. Digoxigenin-11 dUTP signals were detected with brightfield microscopy at 500-fold magnification.


  Results and Discussion
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Materials and Methods
Results and Discussion
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Before RT in situ PCR hybridization, FACS analysis was used to confirm that the cell lines AA2, H9, and JY expressed CD59 and CD55 on the cell surface. All three of them expressed CD59 and CD55 on the cell surface (data not shown). In contrast, on the surface of PBMCs obtained from PNH patients, reduced CD55 expression was observed (data not shown).

Expression of PIG-A mRNA in AA2, H9, and JY Cells
Strong positive signals were observed in all three cell lines using fluorescein, Texas Red, and DIG-11 dUTP (Figure 2A, Figure 2C, and Figure 3A, respectively). Because comparable results were obtained with all three cell lines, only the results of one (AA2) are shown in Figure 2 and Figure 3. Initially, to determine the specificity of our RT in situ PCR assay the following controls were performed: (a) omission of gene-specific primers; (b) omission of the RT step; or (c) use of primers unrelated to the PIG-A gene. Finally, in the experiments presented here the gene-specific primers were omitted in the negative controls. Using this method, we observed the lowest background signals (Figure 2B, Figure 2D, and Figure 3B).



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Figure 2. RT in situ PCR hybridization of PIG-A transcripts in the cell line AA2 using fluorescein labeling (A), negative control (B), and Texas Red labeling (C). Negative control (D). Bar = 20 µm.

Figure 3. RT in situ PCR hybridization of PIG-A transcripts in the cell line AA2 using DIG-11 dUTP labeling (A), negative control (B), RT in situ PCR hybridization of PIG-A transcripts in PBM cells derived from PNH patients using fluorescein labeling (C). Fluorescein labels were detected by simultaneous use of brightfield phase contrast and fluorescence microscopy. Bar = 20 µm.

In the indirect in situ RT PCR assay, a dark-blue precipitate was seen in the cytoplasm and the nucleus (Figure 3A), whereas, in comparison, the negative controls were clearly negative (Figure 3B). As reported by Nuovo 1997 , we observed that the outer membrane of the cells passively absorbed the chromogen, resulting in a "rimmed" background staining.

Analysis of PIG-A Transcripts in PNH Patients
Patients suffering from PNH have one or more mutant hematopoietic stem cell clones that are deficient in GPI anchor synthesis because of somatic mutations in the X-linked gene PIG-A (Kinoshita et al. 1995 ). The somatic mutations of PIG-A are small, varied, and widely distributed in the coding region and splice sites, indicating that they occur at random sites.

PBM cells of two PNH patients were isolated and analyzed for PIG-A gene transcripts by RT in situ PCR hybridization, using A1-B7 fluorescein-labeled oligonucleotides. Here we present the results from one patient, but comparable results were obtained with both of them. As shown in Figure 3C, only approximately 30% of the total cells were specifically stained. These findings correlate well with the reduced PBM cell surface expression of CD55 detected by FACS analysis.

Comparison of the Different Labeling Techniques
Both methods have advantages and disadvantages. The most important advantages of the indirect labeling method are (a) the possibility to use brightfield microscopy and (b) the stability of the labeled amplicon signal. This allows long-term sample storage without loss of signal intensity.

In our hands, the major disadvantages of the indirect labeling method over the direct labeling technique are that the assay is more time consuming and that high dilution of alkaline phosphatase-conjugated anti-DIG antibodies (1:500) requires an overnight incubation for color development, which can enhance the background signals. Moreover, a low dilution of anti-DIG antibodies (1:50) can induce an inadequate precipitate over the cells.

However, the direct method also has some negative aspects. The signal is stable only for a limited time period. Therefore, slides cannot be stored for long periods of time without losing signal intensity. Furthermore, autofluorescence can interfere with the target analysis (Speel et al. 1994 ). For diagnostic purposes, therefore we recommend the use of both the direct and the indirect labeling method in combination, to exclude false-positive or false-negative results.

Finally, fluorescein and Texas Red were also directly incorporated into the PCR-amplified DNA. Interestingly, with this method a less intensive signal was obtained in our experiments. In addition, a smaller number of cells were positively stained.

The concentration of modified nucleotides may be critical for successful labeling (Nuovo 1997 ). An excess of labeled nucleotides may result in a weak or faded signal. This might be explained by an inhibitory effect on the enzyme by the modified nucleotide or by neighboring labels quenching each other. Furthermore, a mixture of labeled and unlabeled cognate nucleotides may be used to improve signal detection. Therefore, decreasing the concentration of nucleotide used or altering the ratio of labeled to unlabeled nucleotides could improve signal detection using direct labeled oligonucleotides.


  Outlook
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Results and Discussion
Outlook
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Multicolor fluorescence in situ hybridization (FISH) has been successfully applied on human chromosomal spreads (Dauwerse et al. 1992 ). In the same manner, it may be possible to use in situ RT PCR to simultaneously detect different transcripts in the same cell. This method could also be used for diagnostic and possibly for therapeutic applications. Recently, Nuovo (1997) reported that RT in situ PCR hybridization can be used to detect a specific single point mutation within the tumor necrosis factor (TNF) gene.

Miyata et al. 1994 analyzed the structure and function of the PIG-A mRNA in patients with PNH. They found mutations within the PIG-A gene transcript that resulted in a nonfunctional PIG-A protein. Yamada et al. 1995 reported that mutations, single base exchanges, single base insertion, frame mutations, and abnormal splicing frequently occurred at random sites in the PIG-A gene in patients with PNH.

To date, hetero-duplex analysis and single-strand conformation polymorphism (SSCP) methods are frequently used to detect single mutations. The combination of one of these two techniques with RT in situ PCR hybridization would be a powerful means of confirming the presence of mutations in PNH or other PIG-A/GPI related disorders.


  Acknowledgments

We thank A. Rentounis for the FACS analysis, K. Zimmermann for helpful comments and suggestions, and S.A. Ali and H.C. Joao for critical reading of the manuscript.

Received for publication April 7, 1997; accepted June 19, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results and Discussion
Outlook
Literature Cited

Boyum A (1974) Separation of blood leukocytes, granulocytes and lymphocytes. Tissue Antigens 4:269-274 [Medline]

Dauwerse JG, Wiegant J, Raap AK, Breuning MH, Van Ommen GJB (1992) Multiple colors by fluorescence in situ hybridization using ratio-labeled DNA probes create a molecular karyotype. Hum Mol Genet 1:593-598 [Abstract]

Kinoshita T, Inoue N, Takeda J (1995) Defective glycosyl phosphatidyl-inositol anchor synthesis and paroxysmal nocturnal haemoglobinuria. Adv Immunol 60:57-103 [Medline]

Miyata T, Yamada N, Iida Y, Nishimura J, Takeda J, Kitani T, Kinoshita T (1994) Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal haemoglobinuria. N Engl J Med 330:249-255 [Abstract/Free Full Text]

Ninomiya H, Abe T, Shichishima T, Terasawa T, Fujita T (1982) Decay-accelerating factor (DAF) on the blood cell membranes in patients with paroxysmal nocturnal haemoglobinuria (PNH): measurements by enzyme-linked immunosorbend assay (ELISA). Br J Haematol 69:81-87

Nuovo GJ (1993) PCR In Situ Hybridization Protocols and Applications. 2nd ed. Philadelphia, New York, Lippincott-Raven

Nuovo GJ (1997) PCR In Situ Hybridization Protocols and Applications. 3th ed. Philadelphia, New York, Lippincott-Raven

Rosse FW (1990) Phosphatidylinositol-linked proteins and paroxysmal nocturnal haemoglobinuria. Blood 75:1595-1601 [Medline]

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Speel EJM, Jasen MPHM, Ramaekers FCS, Hopman AHN (1994) A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. J Histochem Cytochem 42:1299-1307 [Abstract/Free Full Text]

Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T (1993) Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 73:703-711 [Medline]

Wilkinson DG (1992) In Situ Hybridization: A Practical Approach. Oxford, New York, Tokyo, IRL Press-Oxford University Press

Yamada N, Miyata T, Maeda K, Kitani T, Takeda J, Kinoshita T (1995) Somatic mutation of the PIG-A gene found in Japanese patient with paroxysmal nocturnal haemoglobinuria. Blood 85:885-892 [Abstract/Free Full Text]





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