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

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 × 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 × 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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Table 1

PIG-A gene analysis

Primers	Fluorochromes (5′ end-labeled)	Sequences
A1 Sense	Fluorescein	5′-ACAGCCACGACCCTCTTTCACAG-3′
A5 Sense	Texas Red	5′-GATACCAGCTGCATGACAGGGTG-3′
B7 Anti-sense	Fluorescein	5′-CTTCAGTAAGGGAGGTATTCAG-3′
B12 Anti-sense	Texas Red	5′-TCTTACAATCTAGGCTTCCTTC-3′
B13 Anti-sense	—	5′-AATGATATAGAGGTAGCATAAC-3′
A1 Sense	—	5′-ACAGCCACGACCCTCTTTCACAG-3′
B3 Anti-sense	—	5′-GCTGATGTCAGCTCGGTGCTTAC-3′
Primer sets used and length of amplified regions
Primer sets	Amplified region (BP)
Fluorescein A1–B7	550
Texas Red A5–B12	550
(DIG-11 dUTP) A1–B3	300

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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.

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, 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.

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 (Figures 2A, 2C, and 3A, respectively). Because comparable results were obtained with all three cell lines, only the results of one (AA2) are shown in Figures 2 and Figures 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 (Figures 2B, 2D, and 3B).

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.

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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.

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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.

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.