Division of Nephrology and Hypertension, Richard Roudebusch Veterans Affairs Medical Center, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.
deoxyribonuclease I; restriction enzymes
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INTRODUCTION |
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A VARIETY OF MOLECULAR BIOLOGY techniques can be used to detect RNA. Hybridization of nucleic acid probes to complementary RNA after electrophoretic separation is used extensively in the analysis of gene structure, expression (33), and diagnostic tests (8, 29). These techniques require technically demanding generation of the antisense RNA probe and denaturing gels that may contain harmful chemicals such as formaldehyde. Additionally, these methods do not allow the precise cellular localization of the RNA target. In situ hybridization (ISH) permits the localization of specific nucleic acid sequences at the cellular level and detection of genes expressed at low levels (4). However, the method, though highly specific, may often be overshadowed by relatively low-detection sensitivity (4, 15).
Amplification of target mRNA by PCR is used to enhance sensitivity, enabling localization of low-abundance mRNA in a given cell (1, 4, 5, 10, 20, 21, 25, 26, 30, 31). Various detection methods have been used after target mRNA amplification in tissue sections or cell suspensions (4, 9, 13, 17, 20, 24, 30, 32). RT-PCR followed by a separate ISH step (7, 16) using either a radiolabeled or nonisotopically labeled complementary probe for detection and localization of target mRNA is quite cumbersome. Alternatively, direct incorporation of a labeled deoxynucleotide in the amplification step can be used (2, 4, 10, 15, 24). However, direct incorporation of the labeled dNTP results in a high background from nonspecific amplification after mispriming (extension of primers annealed to nontarget sequences) (14, 23). Hot-start PCR (18) and the use of labeled primers in PCR (6) instead of direct incorporation of labeled deoxynucleotide helps to eliminate background due to mispriming or due to primer-independent events. The use of labeled primers across the intron-exon junctions may also circumvent the problems of nonspecific amplification from genomic DNA (12, 13). In spite of DNase pretreatment of the samples (7, 15, 19, 32) and hot-start modification (18), nonspecific amplification from undigested genomic DNA still remains a severe drawback in presently available methods (11), especially in cases where genomic sequence data is incomplete.
In the present study, we used in situ RT-PCR (9) in a highly modified version. We have addressed the problem of nonspecific background amplification by pretreatment of samples with tetracutter restriction enzymes in conjunction with DNase before RT-PCR. This novel approach coupled with the use of Cy5-labeled sense primer in the PCR step allows identification of target mRNA by fluorescence microscopy. Without compromising sensitivity, our method increases specificity, is rapid, and allows image analysis of confocal microscopic images. Analysis and rough estimation of accumulated, labeled cDNA can be achieved by quantitative microscopy methods as well.
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MATERIALS AND METHODS |
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Materials. Paraformaldehyde was purchased from Sigma (St. Louis, MO). Probe-On Plus slides were purchased from Fisher Scientific (Itasca, IL). Proteinase K tritirachium album was purchased from Amresco (Solon, OH). RNase-free DNase I and biotin-16-dUTP were purchased from Boehringer Mannheim (Indianapolis, IN). RQ1-DNase, RNasin, dNTPs, and restriction enzymes were purchased from Promega Scientific (Madison, WI). Cy5-conjugated streptavidin was purchased form Jackson ImmunoResearch Labs (West Grove, PA). Primers were synthesized by Operon Technologies (Alameda, CA). Superscript II RT enzyme kit was purchased from GIBCO BRL (Rockville, MD), and AmpliTaq DNA polymerase was purchased from PE Biosystems (Foster City, CA).
Tissue fixation and sectioning.
Whole embryonic kidneys or adult murine kidney sections were washed in
cold RNase-free PBS. Tissues were fixed in 4% paraformaldehyde-sucrose at 4°C for 18 h. Fixed tissues were mounted in optimum cutting temperature compound (Polysciences, Warrington, PA), and 5- to 7-µm
cryosections were mounted and placed onto RNase-free Probe-On Plus
slides. The slides were kept at 80°C until used for direct in situ
RT-PCR.
Tissue processing. Before proteinase K digestion, tissue sections were incubated for 10 s at 105°C on a heat block to ensure tissue adhesion to the slides. The slides were then immersed at room temperature for 27 min in proteinase K solution at a final concentration of 6.66 µg/ml. Tissue digestion conditions were standardized by performing digestions in graded concentrations of proteinase K for a fixed time. For standardization of digestion conditions, the highest concentration of the enzyme, which did not change the histoarchitecture of the renal tubules, was used. Initially, digestions were monitored under a light microscope at 2- to 3-min intervals until an optimal digest time was achieved. Enzyme digestions were stopped by incubating the slide for 2 min at 105°C on a heat block. Samples were rinsed briefly, first in PBS and subsequently in diethyl pyrocarbonate (DEPC)-treated water. The slides were air-dried.
After proteinase K treatment, the genomic DNA in each sample was digested in situ using a humidified chamber at 37°C for 3 h with 10-20 units of Sau96I alone or in combination with a tetracutter enzyme (e.g., HaeIII or HpaII) in universal buffer containing 10 units of RNasin in a total volume of 20 µl. The slides were washed for 10 s each in PBS and DEPC-treated water. To ensure complete digestion of genomic DNA in the tissue sections, samples were incubated overnight in a humidified chamber at 37°C with 10 units of RNase-free DNase (1 U/µl final concentration). The slides were then rinsed twice for 10 s each with DEPC-treated water.In situ RT reaction. Tissue sections were overlaid with 10 µl of RT mix, consisting of 1× first-strand buffer (GIBCO BRL), 1 mM each of dATP, dCTP, dGTP, and dTTP, 10 units of RNasin, 6 mM dithiothreitol, 0.5 µM of 3' primer, and 5 units of SuperScript II RT enzyme (GIBCO BRL), and incubated at 42°C in a humidified chamber for 1 h. RT was omitted from the RT mix for RT minus control slides. RT reaction was stopped by incubating the slides for 2 min at 92°C in an MJR PTC-100 thermal cycler (MJ Research, Watertown, MA) fitted with a slide holder. The coverslips were removed, and samples were washed twice with DEPC-treated water.
In situ PCR. For amplification of the target krtk (Tyro3) sequence, PCR was carried out in situ on the sections using an MJR PTC-100 thermal cycler. Slides were kept at 4°C before the start of the PCR reaction. PCR mix (10-20 µl) was overlaid on the sections, and the slide was sealed with adhesive coverslips (Sigma, St. Louis, MO). Two protocols were used, both of which labeled kidney sections specifically with equivalent efficacy. In the first method, reactions were performed in the presence of 1× GeneAmp PCR buffer containing 1.5 mM MgCl2 (PE Biosystems), 0.25 mM dNTP, 1.25 µM 5' forward and 3' reverse primer, and 0.125 units of AmpliTaq DNA polymerase. The 5' primer was conjugated to Cy5. In the second approach, reaction conditions were the same except biotin-conjugated dUTP was added at a concentration of 0.05 mM, and unconjugated 5' primer was used instead of Cy5-conjugated primer.
PCR mix was added to the center of the coverslips (Probe-Clip Press-Seal incubation chamber) as spherical droplets, and tissue sections were placed over the droplets. The chamber consisted of a self-sealing silicone gasket along the circumference of the coverslip. Better sealing was ensured with a colorless nail polish. Cycling reactions were done using hot-start conditions by warming the slide holders to 90°C before placing the glass slides in the slide holders for cycling. PCR was carried out for one cycle at 92°C for 90 s, followed by 30 cycles with denaturation at 94°C for 30 s, annealing at 50°C for 1 min, and extension at 72°C for 1 min. When the PCR was complete, samples were kept at 4°C. Coverslips were removed, and the samples were heated to 92°C for 1 min. Subsequently, slides were soaked for 5 min in 1× PBS at room temperature and counterstained with hematoxylin. Alternatively, in samples in which biotin-conjugated dUTP was used for labeling, the specimens were incubated at room temperature for 30 min with 1 µg/ml of Cy5-conjugated streptavidin. Samples were then washed twice for 2 min at room temperature in 1× PBS and then counterstained with hematoxylin. Samples were overlaid with Permount (Fisher Scientific) and covered with coverslips.Light microscopy. Samples were imaged with a Zeiss LSM 510 confocal microscope equipped with argon and helium/neon lasers. Samples were excited at 633 nm of light, and images were collected with a 650-nm emission filter in the light path. All images were collected using standardized laser intensities and photomultiplier tube settings for amplification and dark levels. All images were processed with Adobe Photoshop on a Micron Millennium computer. Photomicrographs were printed on a Kodak XLS 8500 dye sublimation printer.
Image analysis. Photomicrographs were collected in TIFF file format. Image intensity values were measured using the Metamorph image processing program (Universal Imaging, West Chester, PA) running on a Dell Optiplex computer. In all the image analysis studies, data were collected using standard size and shape regions of interest. Average intensity values of each region of interest were collected and used for data analysis.
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RESULTS |
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Tyro3 is a receptor tyrosine kinase that is highly expressed in
mouse brain (28). This gene is also expressed in murine kidney and was identified while screening differentially expressed kidney transcripts (unpublished observations). Northern blot analysis confirmed that Tyro3 is expressed in brain, lung, thymus,
testes, ovaries, whole embryo, spleen, kidney, and heart tissue
(Fig. 1A). Additionally, Tyro3
expression was greater in S1 renal epithelial cells compared with the
distal convoluted tubule (DCT) epithelial cells (Fig.
1B). To determine the spatial distribution of Tyro3 in
tissue sections, a direct label RT-PCR method was developed utilizing
fluorescent labels that permit direct visualization of transcript
expression by light microscopy. This method has been optimized to
achieve the highest possible signal-to-noise ratio between negative
controls and samples containing RNA transcripts. An important feature
of the negative control images is the utilization of Cy5 as the
fluorophore. In preliminary experiments, significant autofluorescence
was observed when samples were imaged with filter sets optimized for
fluorescein or rhodamine fluorescence (Fig. 2, A and B). At
increasingly red-shifted excitation wavelengths, less autofluorescent
signal was detected. With filter sets optimized for Texas red or Cy3, a
detectable emission signal was still noted in unstained kidney sections
(data not shown). However, minimal fluorescence was observed in images
acquired using 580-excitation and 650-emission filter sets (Fig.
2C). At this wavelength, there were dramatic
improvements in the signal-to-noise ratio.
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In Fig. 3, expression of krtk (Tyro3) in
murine kidney sections is shown. Little staining is noted in samples
imaged without a RT reaction (Fig. 3B). When the in situ PCR
reaction was performed without one of the primers (data not shown) or
Taq polymerase, no staining was detectable (Fig.
3C). In contrast, when direct in situ PCR was performed with
all necessary enzymes, RT and Taq polymerase, diffuse
cytoplasmic staining is observed in proximal tubule segments (Fig. 3,
D and E). Note, however, that the nuclear compartment is relatively devoid of staining. These samples were treated with a tetracutting DNA restriction enzyme, Sau96I,
and subsequently with RNase-free DNase I. In tissue samples in which the restriction enzyme digestions were omitted, significant additional fluorescent signals were detected both in the nuclear compartment and
in the cytoplasm, suggesting that genomic DNA contributed to the
images (Fig. 3F). This effect is clearly demonstrated
in samples labeled with primers for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). In Fig. 3G, the expression of GAPDH
is clearly cytoplasmic in the restriction and DNase-digested sample. In
contrast, samples digested with DNase I alone had significant
fluorescent signal in the nucleus and cytoplasm (Fig. 3H).
In samples treated with a tetracutter restriction enzyme and
subsequently with RNase-free DNase I, and where an RT reaction was
omitted, no signal was detected (Fig. 3B). This important
control confirms that no genomic amplification takes place in the
samples.
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A rough correlation can be drawn from the photomicrograph obtained from direct RT-PCR. In Fig. 3E, the relative amount of Tyro3 expression in proximal tubules (Fig. 3E, areas labeled P) is higher than the amount of expression perceived in distal tubule segments (areas labeled D). This agrees with the Northern blot results shown in Fig. 1B. Direct in situ RT-PCR performed on liver sections yields markedly diminished cytoplasmic staining compared with images collected in comparable kidney sections (Fig. 3I compared with Fig. 3E). This result is also in agreement with the Northern blot data of Fig. 1. By densitometry of the Northern blot, Tyro3 expression in S1 cells was 1.8× that of DCT cells. Image analysis demonstrated that average signal intensities from proximal tubule segments were 1.6× that of distal tubule segments (data not shown). This is in good agreement with the Northern blot data and suggests that with additional refinements, direct in situ RT-PCR may be useful as a semiquantitative tool to determine relative mRNA expression.
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DISCUSSION |
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In the original description of in situ RT-PCR, transcript detection required an ISH step with radioactive probe (31). The resultant X-ray micrograph was overlaid onto a photomicrograph to correlate transcript expression with tissue localization (31). This is a technically demanding procedure, which has limited the utility of this method. Besides, the long incubation time required with radiolabeled probes renders the method unsuitable for prospective clinical diagnosis (3). The direct fluorescence-labeling method outlined in this communication eliminates the ISH step. At the end of this labeling method, the fluorescent signal is directly imaged in the tissue section. Fluorescent signals are simultaneously colocalized with phase-contrast photomicrographs when hematoxylin is used to counterstain tissue sections. An additional advantage of direct in situ RT-PCR described in this paper is the improved signal-to-noise ratio of labeled specimens compared with negative controls or unlabeled samples.
Increased signal strength and signal specificity was achieved by overcoming two obstacles. First, kidney tissue sections have significant autofluorescence when imaged with excitation and emission filters optimized for fluorescein and rhodamine. We found that endogenous fluorescent signal was markedly attenuated at longer emission wavelengths. At an emission wavelength of 645 nm, which is the emission maximum for Cy5, tissue sections had little to no detectable endogenous fluorescence. We also found that Texas red could be used as an alternative fluorescent probe, although at this emission wavelength (545 nm), some background fluorescence was observed. Reduced background fluorescence improved the interpretation of fluorescence signals and analysis of signal specificity.
The second problem we identified was the contribution of residual genomic DNA to the images. Intense nuclear staining was observed in samples without prior DNase I treatment. DNase I pretreatment of samples (7, 15, 22, 32) did not eliminate nonspecific amplification from undigested genomic DNA (11). In our experiments, DNase I treatment alone did not eliminate nuclear labeling entirely. To further improve the specificity of labeling, we analyzed the DNA sequence of ktrk (Tyro3) between the PCR primers and identified restriction enzymes that had multiple potential cut sites. Sau96I, a tetracutter restriction enzyme that specifically cuts within the krtk sequence delineated by the primers, was chosen to completely digest genomic DNA. Sau96I has >10 potential cut sites in the ktrk sequence bracketed by the PCR primers (data not shown). Adding a specific restriction enzyme digestion step to the experimental protocol, followed by DNase I digestion, completely eradicated nuclear staining from samples in which a RT reaction or RT-PCR reaction was performed. Further evidence that restriction digestion eliminated the possibility that genomic DNA contributed to our results can be seen by the absence of staining in renal glomeruli (Fig. 3E). When choosing a tetracutter restriction enzyme to digest genomic DNA, we found that restriction enzymes that cut within the DNA sequence bracketed by the chosen PCR primers appeared to work better than enzymes that did not specifically cut within the targeted genomic DNA sequence (data not shown).
Direct incorporation of a labeled deoxynucleotide in the amplification step has been used for detection of mRNA in cells or tissue sections (2, 3, 10, 15, 24). However, direct incorporation of the labeled dNTP has been reported to result in a high background from nonspecific misprimed amplification (14, 23). The use of Cy5-labeled 5' primer in the PCR step eliminates this potential problem very effectively. However, we did not observe any significantly higher background in samples with direct incorporation of biotinylated dUTP (Fig. 2F) that were subsequently detected with Cy5-labeled streptavidin. This may have been possible due to the additional restriction digestion step before DNase treatment of the samples.
The improved specificity of direct in situ RT-PCR and the elimination of a time-consuming ISH step in this protocol should extend the utility of this method. Potentially, direct in situ RT-PCR may be employed to extend the accuracy of cancer diagnosis in biopsy specimens. Additionally, because fluorescent signals are easily quantified, it is possible to automate sample processing and identification of positively labeled specimens.
In summary, we have developed a method of in situ RT-PCR that is highly specific and enables investigators to identify the sites of RNA transcript production in tissues. This method eliminates the ISH step found in prior descriptions of in situ RT-PCR (6, 27). This improvement in mRNA detection and relative ease of sample preparation may increase researcher interest in this method and may possibly be used for pathological diagnosis.
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ACKNOWLEDGEMENTS |
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R. Bacallao is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-46883 and PO1- DK-53465-01. A Clinician Scientist Award to R. Bacallao from the National Kidney Foundation supported this work. R. Kher is a recipient of an American Heart Association fellowship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Bacallao, Div. of Nephrology and Hypertension, Richard Roudebusch Veterans Affairs Medical Center, Indiana Univ. School of Medicine, Indianapolis, IN 46202 (E-mail: rbacalla{at}iupui.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 December 2000; accepted in final form 19 March 2001.
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