ARTICLE |
Correspondence to: Hannu V. Sariola, Institute of Biotechnology, PO Box 56, 00014 University of Helsinki, Finland. E-mail: hannu.sariola@helsinki.fi
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Summary |
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We report an artifactual in situ hybridization (ISH) labeling pattern in embryonic rat tissues. It is caused by a short multiple cloning site-derived sequence incorporated into the RNA probes by in vitro transcription of templates cloned into pBluescript or its descendants. The artifact was seen in tissues in which programmed cell death (apoptosis) takes place during embryogenesis, i.e., in the mesonephric area, developing nervous system, interdigital mesenchyme of the hand plate, and permanent kidney. Labeling of the radioactive ISH with TUNEL verified the co-localization of the artifactual hybridization signal with cells at early stages of apoptosis. Even though the identity of the hybridization target in apoptotic cells remains unknown, it might be highly species-specific, because this artifact was never observed in mouse tissues. (J Histochem Cytochem 48:955961, 2000)
Key Words: in situ hybridization, artifactual signal, rat embryo, pBluescript vector, apoptosis
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Introduction |
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In situ hybridization (ISH) is a powerful technique to visualize the transcription of specific genes at their natural tissue location. The most sensitive way to perform ISH is to use labeled antisense RNA probes that are produced by in vitro transcription of the gene of interest (
The interpretation of ISH labeling is critically dependent on appropriate controls. A widely used method is to transcribe the probe sequences also in the sense direction to provide a control probe with identical hybridization parameters as in the antisense probe. Thus, the sense control is believed to reveal unspecific interactions of the probe with tissues. The template sequence is often cloned into a multiple cloning site (MCS) of a plasmid vector flanked with two distinct promoters enabling the synthesis of sense and antisense probes by different RNA polymerases. The length of the transcript is limited by linearizing the plasmid from restriction sites in MCS downstream to the insert. Short sequences resulting from transcription of the vector itself, between the RNA promoter and insert, are always incorporated into the probe. These synthetic sequences are commonly considered to be insignificant for the hybridization.
We have used radioactive ISH as a routine method, which has provided satisfactory results with more than 100 probes over several years. However, sections from rat embryos have occasionally displayed confusing signals. Therefore, we have now analyzed these experiments and identified a short sequence of the pBluescript (Stratagene; La Jolla, CA) MCS that is responsible for the artifactual hybridization pattern. In embryonic rat samples, this artifact is associated with the regions of apoptosis. By double detection of radioactive ISH and TUNEL, we were able to co-localize the cells hybridizing with the multilinker sequences and those at early stages of apoptotic death.
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Materials and Methods |
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Animals, Tissues, and Preparation of Sections
Pregnant SpragueDawley rats were sacrificed and the embryos were dissected in Dulbecco's phosphate-buffered saline (PBS). The day after overnight mating was considered as the Day zero of embryogenesis (E0). E13 embryos were fixed overnight in 4% paraformaldehyde (PFA) in PBS at 4C and E13 urogenital blocks and E15 kidneys for 2 hr at room temperature (RT). Developmental stage-matched samples were also collected from CBA x NMRI mice (plug day was E0). The embryos and tissues were mounted in paraffin after ethanol series and 7-µm sections were cut to silane-coated glasses.
Plasmids
The pBluescript KS+/- and SK+ vectors were from Stratagene and pGEM3Zf(+) from Promega (Madison, WI). The MCS of pBluescript KS+ was deleted by KpnI-SacI digestion and the ends blunted with T4 polymerase (Promega) and joined via intramolecular ligation, which created a new MscI restriction site between the T3 and T7 promoter. The resulting plasmid was named pBluescript KS. The pBL-KA15 plasmid (a gift from Dr. Nagata, Japan) contained a full-length cDNA of mouse FasL inserted into the XhoI site of pBluescript KS+ in antisense orientation from the T3 promoter. The pMF-1 plasmid (also a gift from Dr. Nagata) contained a full-length murine Fas cDNA inserted into the EcoRI site of pBluescript KS+ in sense orientation from the T3 promoter. Sequencing and restriction enzyme digestions verified the orientation of the template sequences and the identity of the plasmids.
Preparation of [35S]-cRNA Probes
The plasmids used as templates were purified with a maxiprep kit (Qiagen; Hilden, Germany) according to guidelines given in the company's manual. The plasmids were fully linearized with suitable restriction enzymes overnight and after reaction the restriction mixtures were phenolchloroform-extracted and ethanol-precipitated. The linearized plasmids were then in vitro transcribed in the presence of [35S]-UTP. The reaction mixture consisted of 1 x transcription buffer (Promega), 1 µg template plasmid/reaction, 0.2 M DTT (Promega), 2.5 mM GTP/ATP/CTP (Promega), 50 µCi [35S]-UTP (>1000 Ci/mmol) (Amersham Pharmacia Biotech; Uppsala, Sweden), 20 U of rRNase inhibitor (Promega), and 1520 U of appropriate RNA polymerase (Promega). The reaction volume was adjusted to 20 µl with nuclease-free water (Promega). After incubation (37C for 1 hr) 0.5 U of RQ1 RNase-free DNase (Promega) was added and incubation was continued for 30 min. The transcription product was purified by a nick column (Amersham Pharmacia Biotech). From 400 µl fractions of the eluate [10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM DTT, 0.1% SDS], a 0.5 µl sample was taken and its radioactivity was measured with a scintillation counter (LKB Wallac; Turku, Finland). Only the labeling products with the peak activity in the second fraction were used for the subsequent hybridization step. We routinely discarded weakly labeled probes (total activity less than 8.0 x 107 cpm). The high-activity fraction was precipitated with 5 M NaAc (pH 5.2) and absolute ethanol overnight. The precipitated RNA was washed with 70% and absolute ethanol, air-dried, dissolved in a small amount of hybridization buffer [60% deionized formamide (Riedel-de Haën; Seelze, Germany), 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 10% dextran sulfate (Sigma; Natick, MA), 1 x Denhardt's solution (Sigma), 0.5 mg/ml yeast RNA (Sigma), 100 mM DTT (Promega)] and diluted to a final concentration of 2.0 x 104 cpm/µl. During the dilution process the probes were kept on ice and denatured for 2 min at 80C before application to the slides.
In Situ Hybridization
ISH was performed as described by
After hybridization the glasses were washed with 5 x SSC, 10 mM DTT at 50C for 30 min. The high-stringency wash was performed with 50% deionized formamide, 2 x SSC, 30 mM DTT at 65C for 30 min. After three 10-min washes with NTE buffer [0.5 M NaCl, 10 mM Tris-HCl (pH 8.0), 5 mM EDTA], the samples were treated with 20 µg/ml ribonuclease A (Roche; Basel, Switzerland) in NTE buffer at 37C for 30 min and washed with buffer once more. Then the high-stringency wash was repeated. The glasses were washed with 2 x SSC and 0.1 x SSC at 37C for 15 min and dehydrated in ethanol series (30%, 60%, 80%, 95% ethanol) with 0.3 M ammonium acetate. After two washes with absolute ethanol, the glasses were air-dried.
Autoradiography
The dried glasses were dipped in NTB2 autoradiographic emulsion (Kodak; Paris, France) diluted 1+1 in deionized water containing 2% glycerol. After air-drying of the emulsion, the glasses were packed in light-tight boxes with silica gel and exposed at 4C for 23 weeks. The slides were developed with D-19 developer, fixed with sodium fixative (Kodak), and counterstained with hematoxylin (Shandon; Pittsburgh, PA), then dehydrated and mounted in Mountex (Histolab Products; Västra Frölunda, Sweden).
Labeling of Radioactive ISH with TUNEL
The sections hybridized as described above were developed, fixed, and TUNEL staining by the ApopTag In Situ Apoptosis Detection Kit (Intergen; Purchase, NY) was performed according to the manufacturer's instructions. The slides were then washed with PBS twice for 15 min and treated with equilibration buffer for 1 min. Then the reaction mixture was pipetted onto the sections and covered carefully with a plastic coverslip. The terminal transferase reaction was performed at 2830C for 3 hr to avoid melting of the autoradiographic emulsion. The reaction was stopped by a wash solution for 30 min at RT. The slides were washed twice with PBS and mounted in Immumount (Shandon). The samples were photographed with an Olympus Provis AX70 microscope (Olympus Optical; Tokyo, Japan) with brightfield, darkfield, and phase-contrast optics and fluorescence microscopy.
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Results |
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A distinct ISH pattern in embryonic rat tissues was found to be artifactual because it was not dependent on the orientation of the template sequences but rather on the identity of the plasmid used and the direction of the transcription. Furthermore, this hybridization pattern was never detected in embryonic mouse tissues of matching age. When the probe sequences were transferred to the pGEM3Zf(+) vector (Promega), the signal artifact was no longer seen (not shown).
To ascertain that the hybridization signal was derived from pBluescript sequences, we performed a series of ISH experiments with probes transcribed from the T3 promoter of "empty" pBluescript vectors linearized with BamHI (Fig 1). The artifactual signal was detected with pBluescript KS+ and KS- (MCS in KpnI SacI orientation between T3 and T7), but not with pBluescript SK+, in which MCS is in the SacI
KpnI direction between T3 and T7. In addition, the MCS-deficient pBluescript
KS did not produce any signals. The hybridizing sequence was therefore localized to the KpnI end of the MCS. A probe produced from the T3 promoter of pBluescript KS+ linearized with HindIII (not shown) further limited the sequence between KpnI and HindIII restriction sites in the pBluescript MCS.
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We searched the vector database (www.ncbi.nlm nih.gov/VecScreen) with the 36-base KpnI-HindIII oligonucleotide sequence using BLAST (
The artifactual ISH signal was observed in the E13 rat embryo in tubular structures and stromal cells of the posterior mesonephros (Fig 2A and Fig 2B). In addition, the mesenchyme surrounding the notochord (Fig 2C and Fig 2D) and single cells in the developing nervous system were labeled (Fig 2E2G). At E15, the artifactual hybridization signal was seen in the forelimbs at the distal and central parts of the interdigital mesenchyme (Fig 2H2J) and in the metanephros, where strong punctate signals were observed in the renal medulla (Fig 3A and Fig 3B). Routine hematoxylin counterstaining revealed typical apoptotic cells, with nuclear pyknosis and fragmentation, in all the above-mentioned locations (Fig 2G). By combining TUNEL staining with ISH, the artifactual in situ signal in the metanephric kidney was shown to co-localize with TUNEL-positive cells with large, diffuse nuclei (Fig 3A3F).
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Discussion |
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We report here that a short sequence in the multilinker of pBluescript cloning vector hybridizes with a characteristic pattern to embryonic rat tissues. By comparing the results from specific restrictions of pBluescript vectors, we show that the region between KpnI and HindIII in the MCS is responsible for the false hybridization signal. Previously, pBluescript vector multilinker sequences have been shown to bind with the residual bodies of the stage IX and X seminiferous tubules in rat testis (
The artifactual ISH signal was seen in tissues in which extensive apoptotic death is taking place during normal embryonic development. In E13 rat embryos, the vestigial embryonic kidney, the mesonephros, undergoes drastic regression starting from the posterior mesonephric elements (
Most cell death events during development are morphologically recognizable as apoptosis (
This ISH signal is particularly cumbersome to identify as an artifact. First, it is obviously highly species-specific and is never detected with embryonic mouse tissues. Second, the signal is immediately detected as false only if KpnI-HindIII sequence is incorporated into the sense probe. If it is part of the antisense probe, the negative sense control may lead to misinterpretation of the artifactual signal as a natural distribution of the target mRNA. The most deceptive combinatorial signals will be achieved when an antisense probe mixes the true expression pattern of the gene and the artifact. Third, the artifact is not dependent on the identity of the promoter used (T3 vs T7) per se, because the pBluescript is available as two versions containing the MCS in opposite directions (KS vs SK). Whether the KpnI-HindIII sequence is part of the sense or the antisense probe is dependent on both the version of the plasmid and the direction of the transcription. Finally, several different Stratagene vectors harbor the pBluescript MCS sequence causing the artifactual signal. Therefore, if the probes are intended for ISH of rat tissues, the pBluescript family of vectors should be avoided.
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Acknowledgments |
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Supported by the Sigrid Jusélius Foundation and by the Academy of Finland.
We wish to thank Dr Shigekazu Nagata (Osaka Bioscience Institute; Osaka, Japan) for Fas and FasL probes. Ms Marja-Leena Peltonen and Ms Alla Hanninen are acknowledged for skillful technical assistance.
Received for publication February 4, 2000; accepted February 9, 2000.
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