ARTICLE |
Correspondence to: Anton K. Raap, Lab. for Cytochemistry and Cytometry, Dept. of Molecular Cell Biology, Leiden Univ. Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands..
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Summary |
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With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1 and ß-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH. (J Histochem Cytochem 46:12491259, 1998)
Key Words: mRNA, fluorescence in situ, hybridization, oligodeoxynucleotides, horseradish peroxidase, tyramide, signal amplification, cytokine, gene expression, Northern hybridization, RT-PCR
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Introduction |
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THE HUMAN GENOME contains about 60,00080,000 genes of which, in an average cell, approximately one third are believed to be actively transcribed (
To determine the presence and abundance of an mRNA in a tissue or cell sample, a variety of techniques are available. Northern hybridization and the more sensitive reverse transcriptase polymerase chain reaction (RT-PCR) are most frequently used for this purpose. With appropriate calibration these techniques also allow relative and absolute quantitation of mRNA abundance, and spatial and temporal differences in mRNA expression can likewise be determined. However, they do have the basic limitation that only average mRNA abundance of a tissue or cell sample can be determined, which does not provide data on the frequency and the histo- and cytological characteristics of the mRNA-producing cells.
In situ hybridization (ISH) has the ability to visualize mRNA sequences at the (sub-)cellular level and has therefore become an important additional tool for gene expression studies (
In ISH, the contribution of nonspecifically bound probe to the total amount of signals obtained is assessed indirectly by execution of control experiments with sense probes or probes specific for RNA targets known not to be expressed in the cells under study. In addition, the specific probe should be hybridized to a cell sample in which no expression of the sequence is expected and, finally, mock (no probe) hybridizations will show the level of noise introduced by the various immunological detection layers. All these negative control experiments give an indication of which fraction of the ISH signals in a cell reflects noise signals and which fraction represents genuine mRNA hybridization signals.
Noise signals derive from two sources: nonspecific probe binding and nonspecific binding of the various immunological detection layers used for visualization. Reduction of probe complexity can, in principle, reduce hybridization noise. However, a decrease in probe size inevitably leads to a decrease in signal generation capacity because a smaller number of labels can be attached to shorter probe sequences. Although restricting the number of antibody layers will keep the immunocytochemical noise to a minimum, this also reduces sensitivity.
Tyramide signal amplification (TSA) has been recently introduced and has proved to considerably increase the signal intensities in various immunocytochemical and FISH applications (
All these aspects concerning the sources of noise signals, the need for reducing them for low mRNA copy number detection, and the signal generation capacity of TSA have led us to develop a new FISH strategy in which oligodeoxynucleotides (ODNs) are directly conjugated to horseradish peroxidase (HRP), the enzyme driving the TSA reaction. In contrast to hapten-labeled ODNs, this direct HRP conjugation allows deposition of tyramides immediately after hybridization and thus avoids TSA amplification of nonspecifically bound HRP-conjugated primary immunoreagents (
Previously, we have demonstrated that mRNA transcribed in abundance from the human cytomegalovirus immediate-early (HCMV-IE) gene cluster of rat 9G cells can readily be detected using a single ODNHRP probe and TSA detection. In addition, the 50 tandem repeat copies of the integrated HCMV-IE gene cluster were detectable using a single ODNHRP and TSA detection in metaphase spreads at high efficiency (
The ODNHRP/TSA approach may therefore have the potential to detect low-abundance mRNA sequences by FISH. The purpose of this study was to test this hypothesis and to assess the sensitivity limits of mRNAFISH with ODNHRP probes and TSA detection. We used the human bladder carcinoma 5637 cell line, which shows a wide range of expression levels of a variety of cytokine genes (
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Materials and Methods |
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Cell Culture
Exponentially growing human bladder carcinoma 5637 and human osteosarcoma U2OS cells were left untreated or were treated with 50 µg/ml cycloheximide (CHX) (Sigma; St Louis, MO). After 4 hr of incubation, cells were washed with PBS and used for total RNA isolation. For FISH studies, cells were cultured on uncoated glass microscope slides (
Semiquantitative Northern Hybridization and RT-PCR
Semiquantitative Northern hybridization was performed according to 32P]dCTP by random priming (Boehringer Mannheim; Indianapolis, IN).
cDNA synthesis and PCR amplification of the specific cytokine cDNA sequences were performed according to
Probes
Cytokine cDNA probes were labeled with digoxigenin-11-dUTP (Boehringer Mannheim) by standard nick-translation. 30-mer 5'-aminohexyl-oligodeoxynucleotides (aminoODNs) were purchased from Eurogentec (Seraing, Belgium) (for nucleotide sequence, position, and genebank accession numbers see Table 1) and checked for absence of human sequence cross-homologies using the BLAST program. The aminoODNs were conjugated to horseradish peroxidase (Pierce; Rockford, IL) using bifunctional crosslinkers and were purified by IE-HPLC as described by
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Just before hybridization, the ODNHRP probes were diluted in hybridization mixture containing 40% deionized formamide, 2 x SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0), 10% dextran sulfate, 10 mM EDTA, and 2 x Denhardt solution to a final concentration of 100 ng/ml.
In Situ Hybridization and TSA Detection
RNAFISH was performed according to
For cytoplasmic and nucleolar staining, a 40-mer 28S ribosomal RNA-specific ODN probe was labeled with Texas Red (28S-TR). After the final immunological detection layer was applied, cells were hybridized with the 28S-TR probe for 20 min at 37C as described above. Cells were washed with 2 x SSC for 5 min at 37C, dehydrated, air-dried, and embedded in Vectashield (Vector Labs; Burlingame, CA) containing 40 ng/ml 4,6-diamidino-2-phenylindol.2HCl (DAPI) for nuclear counterstaining.
Slides were examined with a Leica DM microscope equipped with a single bandpass filter for fluorescein and Texas Red and x40, x63 objectives and a x100 oil objective with 1.3 numerical aperture. Photographs were taken with Scotch 3M 640 ASA color slide film and slides were exposed for 30 sec.
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Results |
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At the beginning of this study we could not predict where the sensitivity limits of the RNAFISH technique would lie. Therefore, we broadened the mRNA level range for methodological purposes by treating the cells with CHX, a protein synthesis inhibitor. The U2OS cell line, which does not express cytokines, was used as a negative cell control throughout this study.
Relative Quantification of mRNA Levels by Northern Hybridization and RT-PCR
Northern hybridizations were performed on 10-fold decreasing amounts of total RNA extracted from untreated and CHX-treated 5637 and U2OS cells. The results are shown in Figure 1.
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Untreated 5637 cells showed distinct interleukin (IL)-1ß and IL-8 mRNA bands after 24-hr autoradiographic exposure, whereas IL-6, granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and IL-7 mRNA were undetectable. After 4 days of autoradiography a faint G-CSF mRNA band was detectable but the IL-6, GM-CSF, and IL-7 remained undetectable. CHX treatment resulted in increased IL-1ß, IL-8, IL-6, and G-CSF mRNA expression. IL-7 mRNA remained undetectable, and GM-CSF mRNA transcripts were detected only after 4 days of autoradiography. U2OS cells showed no cytokine mRNA expression except for low IL-8 expression after CHX treatment. Expression levels of the two housekeeping genes did not change in 5637 and U2OS cells after CHX treatment. The quantitatively less reliable but more sensitive RT-PCR experiments confirmed the Northern hybridization results (see Figure 2). Because of its higher sensitivity, IL-6, G-CSF, GM-CSF and IL-7 mRNAs were detectable in untreated and CHX-treated 5637 cells. The RT-PCR for IL-8 clearly showed the CHX induction of IL-8 mRNA expression in U2OS cells. U2OS cells (CHX-treated or not) were weakly positive for a few cytokine mRNAs with undiluted total cDNA (IL-6, GM-CSF, and IL-7). The Northern hybridzation and RT-PCR studies showed that in untreated 5637 cells cytokine mRNAs can be detected at levels up to 10,000-fold less than an abundantly expressed housekeeping gene such as human elognation factor-1 (HEF-1
). (Quantitative interpretation of the Northern hybridzation and RT-PCR experiments is in Table 2).
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Optimization of mRNAFISH
Initially, we optimized the TSA detection procedure for RNAFISH using either digoxigenin- or HRP-labeled ODN probes specific for IL-1ß or IL-8. The following experimental parameters were tested: (a) the use of direct TSA reagents (fluorescein, rhodamine, and Cyanine3tyramides) and indirect TSA reagents (biotin TSA and biotin
The use of all direct TSA reagents led to an overall intense fluorescent staining of the cells and prevented detection of specific hybridization signals above this relatively high level of fluorescence. This staining could be caused by binding of the charged fluorescent moiety of the direct TSA reagents to the protein-rich cell matrix. Using the indirect biotin TSA reagents, such cytological staining was not observed. When the performances of biotin TSA and biotin TSA plus were compared, it appeared that the use of biotin TSA plus resulted in far more intense signals. Furthermore, TSA development at 37C was superior to development at 4C or 22C in terms of fluorescent intensity of the signals.
On hybridization of digoxigenin-labeled negative control ODN probe and deposition of biotin-TSA plus after anti-digoxigeninHRP incubation, punctate fluorescent dots were observed when either avidinfluorescein or mouse anti-biotinfluorescein was used for visualization. Similar observations were made with cell control experiments. This clear background pattern could not be attributed solely to nonspecific hybridization of the digoxigenin ODN probe, because with mock (no probe control) hybridizations fewer but still clear background spots were observed. In contrast, hybridization of control ODNHRP probes did not lead to the punctate background pattern, and we therefore concluded that the primary mouse anti-digoxigeninHRP detection layer contributes strongly to distinct background signals on cells and glass surface.
We noted that avidinfluorescein caused some nonspecific staining in 5637 cells, but this proved to be cell type-dependent.
Additional variables tested included ODNHRP probe concentration, stringency and time of hybridization, and stringency and time of posthybridization washes. All ODNs were 30-mers with 43% GC content. When the formamide concentration in the hybridization or washing solution was raised above 45% and when hybridization times exceeded 30 min, loss of hybridization signals was observed. This loss is most likely caused by inactivation of the HRP moieties (
These experiments led to the ODNHRP/TSA RNA FISH protocol described in Materials and Methods (see also Figure 3 for a schematic representation).
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Detection of Cytokine mRNA by FISH
5637 cells were probed with digoxigenin-labeled cDNA probes for IL-1, IL-1ß, IL-6, IL-8, and GM-CSF cytokine mRNAs, and hybrids were detected with a conventional two-layer immunological detection system. The most abundant of these cytokine mRNAs, IL-1ß (Figure 4A) and IL-8, showed a heterogeneous expression pattern. The lower-abundance cytokine mRNAs showed no signals that exceeded the autofluorescence levels of the cells.
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To enhance the fluorescence intensity of the IL-1ß mRNA-specific hybridization signals, sheep anti-digoxigeninHRP followed by biotin TSA plus detection was used instead of the conventional detection method (Figure 4B). Although strong signals were obtained, a negative probe control showed a considerable amount of noise signal (Figure 4C). The amount and intensity of the noise signal are such that they would interfere strongly with detection of specific signals of low-abundance mRNAs.
Next, we tested the use of a single ODNHRP probe specific for IL-1ß or IL-8 mRNA. When the hybridizations were followed by the biotin TSA plus detection, clear heterogeneous distribution patterns of these mRNAs were observed (yellow fluorescence in Figure 5A and Figure 5B). The contrast between the specific hybridization signals and the background signal had improved considerably compared to the conventional RNAFISH approach (compare Figure 4A and Figure 5A). Hybridizations performed with negative control ODNHRP (sense and nonspecific) probes revealed a small amount of background signal (Figure 5C).
To assess the accessibility of cells to probes and to accomplish cytoplasmic and nucleolar counterstaining of the cells, in situ hybridization of a Texas Red-labeled ODN probe specific for 28S rRNA was performed after the ODNHRP/TSA mRNA FISH. The cytoplasm and nucleoli of all cells revealed a positive 28S rRNA hybridization signal (red signal in Figure 5AE). To further ascertain that the heterogeneous expression patterns of the interleukin mRNAs were not the result of poor accessibility of a subpopulation of cells to nucleic acid probes and reagents, cells were also hybridized with a probe specific for elongation factor mRNA. After hybridization, all cells revealed hybridization signals of almost equal number and intensity, suggesting that all cells are well accessible to probes and antibodies (result not shown). Therefore, the apparent heterogenous cytokine RNA expression pattern cannot be attributed to methodological pitfalls.
FISH of the lower-abundance cytokine RNAs in untreated 5637 cells (i.e., IL-1, IL-6, GM-CSF, G-CSF, and IL-7 in untreated 5637 cells) using a single ODNHRP and TSA detection resulted in mRNA signals that had either low fluorescence intensity, as illustrated in Figure 6A for G-CSF mRNA, or no signal above background. Therefore, cocktails of ODNHRP probes were used, which led to an increased fluorescence intensity of the hybridization signals of mRNAs already detectable with one ODNHRP. Two of the three mRNAs not detectable with a single ODNHRP could be visualized with cocktails. Figure 6B shows the intensity of G-CSF mRNA after hybridization with a cocktail of two ODNHRP probes and Figure 6C after hybridization with a cocktail of three. IL-7 mRNA could be convincingly detected by FISH only with a probe cocktail of four ODN-HRPs (Figure 6D). GM-CSF mRNA was not detectable with the cocktail of three used. The use of probe cocktails led to an increased amount of background signal. However, this additional contribution to noise was outweighed by the gain in signal (compare Figure 6A and Figure 6C).
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Except for IL-8 hybridizations, no clearly cytokine mRNA-containing U2OS cells were observed, although a few cytokines showed faint RT-PCR bands. The increase in cytokine mRNA levels in 5637 cells induced by CHX treatment, as determined by Northern hybridization and RT-PCR, was confirmed by RNAFISH experiments. In 5637 cells, CHX treatment generally resulted in an increase in fluorescence intensity of the specific hybridization signals and in the number of cytokine RNA-containing cells. GM-CSF mRNA, not detectable in untreated 5637 cell, could clearly be detected in a subpopulation of the CHX-treated 5637 cells. In line with the Northern and RT-PCR data, the IL-8 gene revealed strong induction of expression in a subset of the CHX-treated U2OS cells (Figure 5E). In untreated and CHX-treated 5637 cells, one or two nuclear RNA signals were observed next to clear cytoplasmic signals. These signals most likely represent cytokine transcription sites (indicated by arrows in Figure 5A and Figure 5E).
The RNAFISH results obtained with the ODNHRP/TSA method are summarized in Table 3.
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Discussion |
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To tackle the problem of low-abundance transcript detection by mRNAFISH, we extensively explored the potential of ODNHRP probes and TSA in RNAFISH, hypothesizing that the use of small probes would reduce nonspecific probe binding and that TSA would compensate for the loss of signal generation capacity associated with the use of small probes (
We selected the 5637 cell cytokine expression model in the anticipation that on a cellular basis interleukin expression would be fairly homogeneous. It apparently is not. This complicates correlation of the FISH data with the semiquantitative Northern hybridization and RT-PCR results and precludes determination of the sensitivity of the novel RNAFISH methodology in terms of copy number per cell, although the ODNHRP/TSA FISH methodology has led to mRNA detection that was not possible with other FISH approaches.
The heterogeneous expression patterns of the cytokine mRNAs indicate that a subpopulation of cells in the clonal 5637 cell line is transcriptionally active within a certain time frame. This is also reflected in the number of cells showing nuclear RNA signals. The minority of cells showed only one or two nuclear spots in the absence of a cytoplasmic RNA signal. Apparently these cells had activated a gene just before fixation of the cells. Another population of cells revealed, in addition to the nuclear RNA signals, a cytoplasmic signal, indicating that these cells were already transcriptionally active for some time before fixation. Finally, the largest population of cytokine mRNA-positive cells revealed cytoplasmic signals only, suggesting that these cells had turned off their cytokine gene transcription shortly before fixation.
The mechanisms underlying gene expression are complex. The expression of some genes is strictly correlated with a phase of the cell cycle, whereas the expression of others is strictly regulated in development. In addition to transcription factors involved in regulation of gene transcription, long-range chromatin interactions may play a role in regulation of gene expression. For example, studies of mRNA hybridization patterns of individual cells have been shown that the order of transcriptional activity of globulin genes is regulated in time and is dependent on the presence of a locus control region (
As observed in RT-PCR experiments, U2OS cells showed very low expression of IL-6, GM-CSF, and IL-7. However, these low RNA abundances could not be detected with our FISH methodology, indicating that more sensitive in situ transcript detection is needed. An obvious way to increase sensitivity is to apply repeated TSA rounds. However, this leads to unacceptably high levels of background signal (results not shown), but it also shows that with TSA strategies more sensitive transcript detection should be feasible if noise can be further reduced.
Recently, several new probe designs have been reported that may be of use in reaching the aim of further reduction of nonspecific probe binding in FISH.
Use of polypeptide nucleic acid (PNA) probes has also been advocated recently to improve specificity of hybridization. PNAs form more stable hybrids with DNA and RNA sequences than DNA or RNA probes do. In addition, PNAs have greater discriminatory power because of the greater differences in Tm of perfectly matching and single-base mismatching PNA.DNA or PNA.RNA duplexes (820C) ( light-chain mRNA in lymphoid tissue sections and transcripts containing CTG triplet repeats in human myotonic dystrophy cells. In combination with TSA, PNAs may allow low copy number mRNA detection as well.
In conclusion, we developed and explored a novel RNAFISH technology, based on ODNs and HRP-mediated tyramide signal amplification, that permits detection of transcripts in situ that are not or are only marginally detectable by other means. This ODNHRP/TSA mRNAFISH approach is therefore expected to find broad application in basic cell biology and molecular pathology research.
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Acknowledgments |
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Supported in part by the Dutch Science Organization, Area Medical Sciences (MW-NWO) project no. 900-543-109 and by NEN Life Science Products, Boston, MA. CMH was supported by FWF, Austria, no. J1481-MED.
Received for publication June 26, 1998; accepted June 26, 1998.
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