Nonradioactive In Situ Hybridization Research Articles

Automated ISH for Validated Histological Mapping of Lowly Expressed Genes.

An essential part of the drug discovery and development process in the pharmaceutical industry is to provide a full characterization of cells expressing a given drug target and potential downstream markers in human tissues and in relevant preclinical animal species. This task is best solved by a combination of methods, including histological assessment of target protein and mRNA using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively, as well as non-histology-based methods, such as fluorescence-activated cell sorting (FACS), and single-cell (SCS) or single-nuclei (SNS) sequencing. In reality, this work is often complicated by a combination of low target expression levels and a less than optimal availability of specific reagents for detection. In particular, the ability to specifically detect low-abundance receptor targets using IHC is notoriously difficult, due to a daunting lack of commercially available specific antibodies validated for use in IHC. In the absence of fully validated antibodies and protocols for IHC, the specific detection of target mRNA using ISH is often the only available histological method. A highly sensitive, nonradioactive, automated, and robust ISH method for use on formalin-fixed, paraffin-embedded (FFPE) tissue sections is presented for assessing histological localization of mRNA transcripts of lowly expressed genes.

An optimized protocol for high-throughput in situ hybridization of zebra finch brain.

In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-µm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.

Cellular Expression and Localization of Estrogen Receptor α and Progesterone Receptor mRNA in the Bovine Oviduct Combining Laser-Assisted Microdissection, Quantitative PCR, and In Situ Hybridization

The importance of using techniques that allow the study of pure populations of cells has been increasingly recognized. The authors used laser-assisted microdissection (LAM) in combination with quantitative real-time PCR (qPCR) to assess the relative expression of mRNAs encoding estrogen receptor α (ERα) and progesterone receptor (PR) in the different compartments of the bovine oviduct (epithelium, stroma, smooth muscle coat) during the follicular and mid-luteal phases of the estrus cycle. The localization of receptor mRNA was further studied using non-radioactive in situ hybridization (NISH). A special focus was on whether formalin fixation and paraffin embedding influence the quality and quantity of mRNA obtained from microdissected material. Distinct cyclic changes of the mRNA in the bovine oviduct were observed with elevated levels of PR mRNA transcripts in the epithelium and smooth muscle coat during the follicular phase. The expression of PR mRNA did not vary significantly in the stroma of the bovine oviduct during follicular and mid-luteal phases. In conclusion, the authors found that LAM with qPCR can precisely locate and accurately quantify mRNA expression in specific cell populations from formalin-fixed and paraffin-embedded oviductal tissue.

H19 and IGF2 gene expression in human normal, hyperplastic, and malignant endometrium

We examined H19 and insulin-like growth factor 2 (IGF2) gene expression in normal endometrium (12 cases), hyperplasia (27 cases), and cancer (27 cases) by non-radioactivein situhybridization. H19 was not expressed in the epithelium of normal endometrium, but its frequency of expression was 15% in hyperplastic and 60% in neoplastic epithelium. In stroma cells, H19 frequency of expression was 75% in normal endometrium, 55% in hyperplasia, and 37% in carcinoma. According to the grade of endometrial cancer cell differentiation, H19 showed increased frequency and level of expression in the epithelium from well to moderately and poorly differentiated tissues. Our results indicate that H19 expression in epithelial cells of endometrial hyperplasia and cancer merits further investigation and could be useful as a complementary histopathologic and prognostic marker among other modalities in endometrial cancer. IGF2 expression did not appear useful for diagnostic or prognostic purposes.

Measurement of nitric oxide-related enzymes in the brain by in situ hybridization.

Functional genomic increasingly requires the cell-specific localization of gene expression. The histological analysis of mRNA expression is performed by in situ hybridization (ISH), whereas proteins are detected by immunohistochemistry. ISH allows the rapid detection of any transcript with the same protocol provided that its cDNA is known. This is an advantage compared to immunohistochemistry, which necessitates the raise of specific antibodies and the test of different procedures depending on the antigens to detect. However, ISH is subject to multiple technical difficulties. We have developed a nonradioactive ISH protocol allowing the simultaneous coupling of immunohistochemistry to follow gene expression at the protein level on the same section as the mRNA detection. This chapter describes the neuronal nitric oxide synthase (nNOS), argininosuccinate synthetase (AS), and argininosuccinate lyase (AL) mRNA expression in the rat brain to illustrate the methods allowing the nonradioactive localization of transcripts by ISH, coupled to immunohistochemistry to identify the specific cell types of central nervous system (CNS) on the same section.

Co-expression of somatolactin mRNAs in the pituitary gland of gilthead sea bream.

The expression of mRNAs of the two types of somatolactin which have been found, up to the present, in the pituitary of gilthead sea bream (Sparus aurata) have been analyzed by in situ hybridization (ISH). The method of non-radioactive ISH, which we optimize in this study, uses oligonucleotides labelled in the 5' end with biotin or dioxygenin as probes. This allows simple and double ISH of high specificity and sensitivity to be performed. The distinct somatolactin oligoprobes used present a strong signal fundamentally in the pars intermedia of the pituitary gland. For the first time we present cells that co-express both the forms of mRNA mentioned above. Moreover, after studying three groups of different sizes, produced by asynchronic growth of the gilthead sea bream in industrial cultivation, we did not find qualitative differences in the levels of expression of the somatolactin gene.

Retrieval of mRNA from paraffin-embedded human infant brain tissue for non-radioactive in situ hybridization using oligonucleotides

In situ hybridization (ISH) is used to examine the spatiotemporal distribution of gene expression in a range of tissues. Neuroscience research in human brain tissue requires techniques that can be used in formalin fixed and paraffin-embedded tissue rather than frozen tissue which is recommended, but difficult to obtain. This study presents a method for non-radioactive (DIG) ISH for detecting NR1 gene expression, in human infant brain tissue. We compared three pre-treatment effects, protease digestion, autoclaving (in citrate and Tris/EDTA buffer) and microwaving (in citrate and Tris/EDTA buffer). Tissue had been fixed in formalin for 2–12 weeks. Results were compared for the hybridization and background signal intensities, and tissue morphology. We found that optimum results were obtained using 12-min microwave pre-treatment in Tris/EDTA buffer. This method produced optimum signal to background ratio in infant and adult tissue, preserved tissue morphology, and was suitable for use across a broad range of fixation times.

Differential Expression of Genes Encoding Subthreshold-Operating Voltage-Gated K+Channels in Brain

The members of the three subfamilies (eag, erg, and elk) of the ether-a-go-go (EAG) family of potassium channel pore-forming subunits express currents that, like the M-current (I(M)), could have considerable influence on the subthreshold properties of neuronal membranes, and hence the control of excitability. A nonradioactive in situ hybridization (NR-ISH) study of the distribution of the transcripts encoding the eight known EAG family subunits in rat brain was performed to identify neuronal populations in which the physiological roles of EAG channels could be studied. These distributions were compared with those of the mRNAs encoding the components of the classical M-current (Kcnq2 and Kcnq3). NR-ISH was combined with immunohistochemistry to specific neuronal markers to help identify expressing neurons. The results show that each EAG subunit has a specific pattern of expression in rat brain. EAG and Kcnq transcripts are prominent in several types of excitatory neurons in the cortex and hippocampus; however, only one of these channel components (erg1) was consistently expressed in inhibitory interneurons in these areas. Some neuronal populations express more than one product of the same subfamily, suggesting that the subunits may form heteromeric channels in these neurons. Many neurons expressed multiple EAG family and Kcnq transcripts, such as CA1 pyramidal neurons, which contained Kcnq2, Kcnq3, eag1, erg1, erg3, elk2, and elk3. This indicates that the subthreshold current in many neurons may be complex, containing different components mediated by a number of channels with distinct properties and neuromodulatory responses.

Soluble guanylate cyclase gene expression and localization in rat lung after exposure to hypoxia.

The nitric oxide (NO)-cGMP signal transduction pathway plays an important role in the regulation of pulmonary vascular tone and resistance in pulmonary hypertension. A number of studies have demonstrated that endothelial (e) and inducible nitric oxide synthases (NOS) are upregulated in hypoxia-exposed rat lung. These changes in NOS expression have been found to correlate with the process of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension, and remodeling is increased in the absence of eNOS. In this study, we examined the expression and localization of soluble guanylate cyclase (sGC), the primary receptor for NO, in hypoxia- and normoxia-treated rat lungs. Male Sprague-Dawley rats were exposed to hypoxia (10% O(2), normobaric) or normoxia for 1, 3, 5, and 21 days. The lungs were used for Western analysis of sGC protein, sGC enzyme activity, immunohistochemistry using antiserum against sGC alpha(1)- and beta(1)-subunits, and nonradioactive in situ hybridization (NRISH) using a digoxigenin-labeled sGC alpha(1)-subunit cRNA probe. Western blot analysis revealed a more than twofold increase of sGC protein alpha(1)-subunit in rat lungs exposed to 3, 5, and 21 days of hypoxia, correlating well with sGC enzyme activity. Immunohistochemistry and NRISH demonstrated increased expression of sGC in the smooth muscle cells of the pulmonary arteries and arterioles in the hypoxic rat lungs when compared with normoxic controls. Based on our results, the upregulation of sGC may play an important role in the regulation of smooth muscle tone and pressure in the pulmonary circulation during chronic hypoxia.

PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

The method of in situ hybridization (ISH) using non-radioactive detection systems has become a very important molecular tool in research and diagnosis. However, nonradioactive ISH techniques generally detect only relatively abundant DNA or RNA molecules. The methods of target amplification, which multiply target molecules, or the methods of signal amplification, which increase the sensitivity of visualization, have been introduced for the detection of rare targets. Polymerase chain reaction (PCR) has been employed in most approaches to target amplification. Although the general principle of PCR in situ amplification is simple, its practical methods have revealed poor amplification efficiency and reproducibility as well as target localization. We have evaluated PCR in situ amplification methods to detect single copies of viral DNA with an experimental model of cervical carcinoma cell lines, and compared them with catalyzed signal amplification methods that we have developed using hapten-conjugated tyramide. Both amplification methods demonstrated the capability of detecting a single copy of DNA. In this overview, the applications and limitations of both target and signal amplification are summarized.

Microtubules are Needed for Dispersal ofα-myosin Heavy Chain mRNA in Rat Neonatal Cardiac Myocytes

In some cell types, microtubules are used for transport of mRNA through the cytoplasm to the translation site. The number of microtubules increases during growth of cardiac myocytes, suggesting a functional role exists. Here, we test the need for microtubules to transportα-myosin heavy chain (α-MyHC) mRNA through the cytoplasm of neonatal cardiac myocytes. Theα-MyHC mRNA concentration was assessed by non-radioactivein situhybridization. The relative mRNA distributions were expressed as slopes (m=OD/μm), since optical density declined linearly from the nucleus to the cell periphery. Spontaneously-contracting myocytes displayed a gradual decrease inα-MyHC mRNA away from the nucleus (m=−1.27±0.12 OD/μm). To test whether microtubules were necessary forα-MyHC mRNA dispersal, contraction was first arrested with the Ca2+-channel blocker verapamil (10μm) for 18 h, which aggregated the mRNA perinuclearly. Contractile activity was then resumed by washing out verapamil and using isoproterenol (10μm) in the presence or absence of a microtubule depolymerizing drug, colchicine (3μm). Within 6 h, theα-MyHC mRNA distribution in myocytes with microtubules returned to normal values (m=−1.11±0.14 OD/μm), while cells lacking microtubules maintained a perinuclear mRNA distribution (m−1.50±0.16 OD/μm;P<0.05 from control). Despite this perinuclear pattern of mRNA distribution, the myocytes still produced new myofibrils. These data indicate that microtubules are necessary for dispersal ofα-MyHC mRNA outward from the nucleus. Furthermore, myofibrillogenesis may occur independently of mRNA localization and microtubule organization.

Expression of growth hormone receptor in the bovine mammary gland during prenatal development.

Growth hormone (GH) is known to play a key role in postnatal growth and differentiation. The role of GH and its receptor (GHR) in prenatal development, however, is still controversial. Using reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry we demonstrated the presence of GHR mRNA and protein in bovine mammary glands during fetal development. RT-PCR revealed GHR transcripts in fetal mammary glands from the third to the ninth month of pregnancy. By non-radioactive ISH, GHR mRNA was localized in the glandular epithelium, the surrounding mesenchymal cells, endothelial cells of vessels and in the stratum basale of the epidermis of fetal mammary glands. From the sixth month of fetal life onwards, GHR transcripts were also found in the cytoplasm of adipocytes. Immunohistochemical studies using the monoclonal antibody mAb 263 revealed the same distribution pattern as the mRNA. Our results imply that the growth hormone receptor is distinctly expressed in the immature mammary gland, suggesting that GH is involved in growth and differentiation of the fetal mammary gland.

Nonradioactive In Situ Hybridization: Recent Techniques and Applications.

In situ hybridization (ISH) has become a standard method for the localization of nucleic acid sequences in chromosomes, single cells, and tissue sections. Nonradioactive ISH has not only eliminated the problems associated with radioactive probes but has also achieved a higher degree of resolution. Advances in probe preparation and labeling methods have facilitated the general application of ISH. In combination with immunohistochemistry, ISH can provide histological information on gene activity at the DNA, mRNA, and protein levels. Some nonradioactive ISH can simultaneously detect nucleic acid sequences in the same tissue or in a chromosome spread. Advances in ISH technology, including use of the polymerase chain reaction offer both a high sensitivity allowing detection of low levels of gene expression and the cytological localization of gene sequences.

Improvement of Nonradioactive DNA in Situ Hybridization

With the introduction of microwave pretreatment, the quality of nonradioactive in situ hybridization (NISH) using DNA probes on formalin fixed tissue has significantly improved. Even after microwave treatment, however, there are cases where NISH results remain unsatisfactory. Therefore, we tried to improve NISH by testing other buffer systems as alternatives to the citrate buffer that is routinely applied during microwave pretreatment. By using buffer systems originally designed for immunohistochemistry, we significantly improved our NISH results. Difficult tissue samples were more accessible to NISH using these alternative buffer systems and made the quantitative evaluation easier. These results may also be of interest for combined applications of NISH and immunohistochemistry.

Nachweis des angiogenen Zytokins Vascular Endothelial Growth Factor (VEGF) durch nicht radioaktive In-situ-Hybridisierung

In tumor angiogenesis Vascular Endothelial Growth Factor (VEGF) has an important role due to its target cell specificity. It is expressed by the tumor and effects on a paracrine pathway. To increase the understanding of its regulation, it is necessary to identify those cells releasing VEGF. This can be done by in situ hybridization (ISH). In this paper we present a protocol for non-radioactive ISH for VEGF colonic tissue. With this protocol it is possible to perform hybridization within one day.

Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.

In situ hybridization (ISH) has proven to be a very important molecular tool in research and diagnosis. However, its applicability can be limited by its restricted detection sensitivity. During the last few years, several strategies have been developed to improve the threshold levels for ISH detection by amplification of either target nucleic acid sequences prior to ISH or the detection signals after hybridization procedures. In this overview, we outline and analyze the principles, applications, and limitations of in situ polymerase chain reaction, in situ self-sustained sequence replication, primed in situ labeling (PRINS), and in situ transcription as examples of target amplification methods, and catalyzed reporter deposition using biotinylated tyramine as an approach to signal amplification in ISH. We also provide a detailed, 1-day protocol for non-radioactive oligonucleotide ISH including signal amplification, which is suitable for diagnostic purposes. Furthermore, future directions of ISH including combined strategies of target and signal amplification as well as automation are discussed.

SEX CHROMOSOME DETERMINATION IN EXTRAGONADAL TERATOMAS BY INTERPHASE CYTOGENETICS: Clues to Histogenesis

Teratomas are neoplasms that are composed of tissues from all three germinal layers. The exact histogenetic origin of teratomas, however, is still controversial. In order to gain more insight into histogenesis of extragonadal teratomas (EGTs), the gonosomal status in 13 congenital EGTs was studied by means of interphase cytogenetics using nonradioactive in situ hybridization (NISH) with centromere-specific DNA probes. By use of this technique a direct correlation of cytogenetic results with morphology was possible. In all EGTs analyzed the gonosomal status in tissues derived from the different germinal layers was identical to that of the nontumorous fetal and placental tissue. This was true irrespective of localization, age, histological type, and classification of the EGT. Our results strongly suggest that EGTs arise from pluripotent diploid precursor cells, for example, either premeiotic germ cells that have not yet undergone the first meiotic division or pluripotent ectopic embryonal or extraembryonal cells. Our data do not support the theory of parthenogenetic EGT development, at least in males.

Differential Regulation of Ciliary Neurotrophic Factor (CNTF) and CNTF Receptor α Expression in Astrocytes and Neurons of the Fascia Dentata after Entorhinal Cortex Lesion

Neurotrophic factors have been implicated in reactive processes occurring in response to CNS lesions. Ciliary neurotrophic factor (CNTF), in particular, has been shown to ameliorate axotomy-induced degeneration of CNS neurons and to be upregulated at wound sites in the brain. To investigate a potential role of CNTF in lesion-induced degeneration and reorganization, we have analyzed the expression of CNTF protein and CNTF receptor alpha (CNTFR alpha) mRNA in the rat dentate gyrus after unilateral entorhinal cortex lesions (ECLs), using immunocytochemistry and nonradioactive in situ hybridization, respectively. In sham-operated as in normal animals, CNTF protein was not detectable by immunocytochemistry. Starting at 3 d after ECL, upregulation of CNTF expression was observed in the ipsilateral outer molecular layer (OML). Expression was maximal at around day 7, and at this stage immunoreactivity could be specifically localized to astrocytes in the ipsilateral OML. By day 14 postlesion, CNTF immunoreactivity had returned to control levels. CNTFR alpha mRNA was restricted to neurons of the granule cell layer in controls. Three days postlesion, prominent CNTFR alpha expression was observed in the deafferented OML. A similar but less prominent response was noticed in the contralateral OML. After 10 d, CNTFR alpha expression had returned to control levels. Double labeling for CNTFR alpha mRNA and glial fibrillary acidic protein (GFAP) showed that upregulation of CNTFR alpha occurred in reactive, GFAP-immunopositive astrocytes of the OML. A substantial reduction of CNTFR alpha expression in the deafferented granule cells was transiently observed at 7 and 10 d postlesion. Our results suggest a paracrine or autocrine function of CNTF in the regulation of astrocytic and neuronal responses after brain injury.

Sex Chromosome Determination in Extragonadal Teratomas by Interphase Cytogenetics: Clues to Histogenesis

Teratomas are neoplasms that are composed of tissues from all three germinal layers. The exact histogenetic origin of teratomas, however, is still controversial. In order to gain more insight into histogenesis of extragonadal teratomas (EGTs), the gonosomal status in 13 congenital EGTs was studied by means of interphase cytogenetics using nonradioactive in situ hybridization (NISH) with centromere-specific DNA probes. By use of this technique a direct correlation of cytogenetic results with morphology was possible. In all EGTs analyzed the gonosomal status in tissues derived from the different germinal layers was identical to that of the nontumorous fetal and placental tissue. This was true irrespective of localization, age, histological type, and classification of the EGT. Our results strongly suggest that EGTs arise from pluripotent diploid precursor cells, for example, either premeiotic germ cells that have not yet undergone the first meiotic division or pluripotent ectopic embryonal or extraembryonal cells. Our data do not support the theory of parthenogenetic EGT development, at least in males.

P53-expression and chromosome 17-copy number in colorectal adenomas

To get better insight into the role of numerical chromosome 17-aberrations and functional loss of the tumor suppressor gene TP53 during the early steps of colorectal carcinogenesis we analyzed paraffin-embedded tumor tissue from 58 colorectal adenomas with different histological features for p53-expression by immunohistology (IH; moAb DO1), #17-copy number by interphase-cytogenetics [nonradioactive in situ-hybridization (NISH) with a centromer-specific DNA-probe (D17Z1)], and DNA-ploidy by flow cytometry (FCM) with special emphasis on histopathological correlation. Seven adenomas (12%) showed nuclear p53-immunoreactivity. p53-expression was correlated with villous/tubulo-villous growth pattern (p=0.005) and grade of atypia (p=0.003) or dysplasia (p=0.0018). #17-aneusomy was present in 31% of the cases (29% deletions, 2% gains). In the FCM-analysis, 33% of the adenomas were DNA-non-diploid. p53-immunoreactivity correlated statistically significantly with FCM-non-diploidy (p=0.004) suggesting a role for the TP53-gene in the aneuploidization process. #17-deletions were associated to p53-immunoreactivity (p=0.046) but no correlation was found between FCM-ploidy and #17-copy number. As for loss of a tumor suppressor gene both alleles have to be affected, our data suggest a multistep process of TP53-inactivation. Whether the subgroup of adenomas with p53-expression might progress faster into invasive carcinoma than p53-negative cases remains to be tested.