In Situ Hybridization Method Research Articles

Fluorescence quenching by high-power LEDs for highly sensitive fluorescence in situ hybridization.

Recent technical advances have made fluorescent in situ hybridization (ISH) a pivotal method to analyze neural tissue. In a highly sensitive ISH, it is important to reduce tissue autofluorescence. We developed a photobleaching device using a light-emitting diode (LED) illuminator to quench autofluorescence in neural tissue. This device was equipped with 12 high-power LEDs (30 W per single LED) and an evaporative cooling system, and these features achieved highly efficient bleaching of autofluorescence and minimized tissue damage. Even after 60 min of photobleaching with evaporative cooling, the temperature gain of the tissue slide was suppressed almost completely. The autofluorescence of lipofuscin-like granules completely disappeared after 60 min of photobleaching, as did other background autofluorescence observed in the mouse cortex and hippocampus. In combination with the recently developed fluorescent ISH method using the hybridization chain reaction (HCR), high signal/noise ratio imaging was achieved without reduction of ISH sensitivity to visualize rare mRNA at single copy resolution by quenching autofluorescence. Photobleaching by the LED illuminator was also effective in quenching the fluorescent staining of ISH-HCR. We performed multiround ISH by repeating the cycle of HCR staining, confocal imaging, and photobleaching. In addition to the two-round ISH, fluorescent immunohistochemistry or fluorescent Nissl staining was conducted on the same tissue. This LED illuminator provides a quick and simple way to reduce autofluorescence and quench fluorescent dyes for multiround ISH with minimum tissue degradation.

Novel DNA‐based in situ hybridization method to detect Desmozoon lepeophtherii in Atlantic salmon tissues

The microsporidian Desmozoon lepeophtherii Freeman and Sommerville, 2009 is considered significant in the pathogenesis of gill disease in Atlantic salmon (Salmo salar Linnaeus, 1758). Due to the difficulty in detecting D.lepeophtherii in tissue sections, infections are normally diagnosed by molecular methods, routine haematoxylin and eosin (H&E) stained gill tissue sections and the use of other histochemical stains and labels to confirm the presence of spores. An in situ hybridization (ISH) protocol specific for D.lepeophtherii was developed using DIG-labelled oligonucleotide probes. Diseased Atlantic salmon gills were analysed by ISH, calcofluor white (CW) and H&E. All methods showed high levels of specificity (100%) in their ability to detect D.lepeophtherii, but the sensitivity was higher with ISH (92%), compared with CW (64%) and the presence of microvesicles on H&E stained sections (52%). High levels of D.lepeophtherii spores were significantly associated (p<.05) with the development of D.lepeophtherii-associated pathology in the gills, with Ct values below 19 and over 100 microsporidia/10 mm2 of gill tissue (from the ISH counts) seemingly necessary for the development of microvesicles. The ISH method has the advantage over other histological techniques in that it allows all life stages of the microsporidian to be detected in infected salmon gill tissue sections.

Discordance Between Immunohistochemistry and In Situ Hybridization to Detect HER2 Overexpression/Gene Amplification in Breast Cancer in the Modern Age: A Single Institution Experience and Pooled Literature Review Study

BackgroundHuman epidermal growth factor 2 (HER2) amplification and/or overexpression occurs in 12% to 25% of breast cancers. Accurate detection of HER2 is critical in predicting response to HER2-targeted therapy. Both immunohistochemistry (IHC) and in situ hybridization (ISH) are FDA-approved methods for detecting HER2 status because its protein overexpression is largely attributable to gene amplification. However, variable discordant results between IHC and ISH have been reported. MethodsWe determined the frequency of HER2 IHC/ISH discordance in these patients and also performed a pooled literature review analysis. ResultsOf the 1125 consecutive primary or metastatic breast cancers with HER2 IHC and ISH performed simultaneously between 2015 and 2020, 84.6% had an unequivocal HER2 status. Discordance was found in 30 cases from 26 patients, including 13 IHC−/ISH+ and 17 IHC+/ISH−, representing 1.6% and 11.9% of IHC− and IHC+ cases, respectively. Review of the literature between 2001 and 2020 identified 46 relevant studies, with a total of 43,468 cases with IHC and ISH performed. The IHC−/ISH+ and IHC+/ISH− discordances were seen in all antibody clones and ISH methods used. The IHC+/ISH− discordance was significantly higher than IHC−/ISH+ (13.8% vs. 3%, P < .0001). The overall discordance constituted 4% of all cases and 5.4% of those with an unequivocal IHC status. Significantly lower incongruities for both IHC−/ISH+ and IHC+/ISH− were found in those published after 2018. The discordances probably reflect altered biology of HER2 oncogene/oncoprotein. Routinely performing both IHC and ISH may uncover such cases to prevent denial of potentially beneficial targeted therapy.

Multiplex in situhybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics.

Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcript molecules at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We instead multiplex within a single transcript, using probes targeted to the 5' and 3' regions of muscle dystrophin mRNA. Our approach shows this method can reveal transcriptional dynamics in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs in quantitative fashion (with the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggest this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Demonstration of microRNA using in situ hybridisation on formalin fixed paraffin wax samples using conventional oligonucleotide probes: a comparison with the use of locked nucleic acid probes

ABSTRACT Background MicroRNAs (miRNAs) regulate the translation of mRNA during gene expression and investigations have highlighted their importance in pathophysiology. qRT-PCR is currently the gold standard method for detecting changes in miRNA expression. However, when used on heterogeneous samples, it cannot identify individual cell types harbouring miRNAs. For this, in situ hybridisation (ISH) can be used. ISH methods using locked nucleic acid (LNA) probes give reliable results in formalin fixed paraffin-embedded (FFPE) samples. In this study their use has been directly compared with conventional oligonucleotide probes (COP) for ISH. Methods FFPE samples of colorectal adenocarcinoma, squamous carcinoma of lung and cases of invasive breast carcinoma were used to evaluate COP and LNA methods for the demonstration of miR-126 and miR-205. To demonstrate the utility of the COP method demonstration of miR-21 in 19 Gleason stage 7 prostate biopsy FFPE tissues was also undertaken. The demonstration of miR-21 by ISH in high and low expressing prostate cancer cell lines was also compared with qRT-PCR. Results Similar results were obtained using the COP and LNA ISH methods for the demonstration of miR-126 and miR-205. miR-21 was successfully demonstrated in the prostate cancer samples by COP ISH and expression levels of the miRNA demonstrated in the cell lines corresponded with qRT-PCR. Conclusion This study has shown that simplification of ISH protocols by the use of COPs provides equivalent results to the use of LNA methods and it can be used to precisely identify cells in which miRNAs are expressed.

Hybridization Chain Reaction for Quantitative and Multiplex Imaging of Gene Expression in Amphioxus Embryos and Adult Tissues.

In situ hybridization (ISH) methods remain the most popular approach for profiling the expression of a gene at high spatial resolution and have been broadly used to address many biological questions. One compelling application is in the field of evo-devo, where comparing gene expression patterns has offered insight into how vertebrate development has evolved. Gene expression profiling in the invertebrate chordate amphioxus (cephalochordate) has been particularly instrumental in this context: its key phylogenetic position as sister group to all other chordates makes it an ideal model system to compare with vertebrates and for reconstructing the ancestral condition of our phylum. However, while ISH methods have been developed extensively in vertebrate model systems to fluorescently detect the expression of multiple genes simultaneously at a cellular and subcellular resolution, amphioxus gene expression profiling is still based on single-gene nonfluorescent chromogenic methods, whose spatial resolution is often compromised by diffusion of the chromogenic product. This represents a serious limitation for reconciling gene expression dynamics between amphioxus and vertebrates and for molecularly identifying cell types, defined by their combinatorial code of gene expression, that may have played pivotal roles in evolutionary innovation. Herein we overcome these problems by describing a new protocol for application of the third-generation hybridization chain reaction (HCR) to the amphioxus, which permits fluorescent, multiplex, and quantitative detection of gene expression in situ, within the changing morphology of the developing embryo, and in adult tissues. A detailed protocol is herein provided for whole-mount preparations of embryos and vibratome sections of adult tissues.

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.

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.

Clinical significance of BRCA1 and BRCA2 mRNA and protein expression in patients with sporadic gastric cancer.

The purpose of the present study was to investigate the clinical significance of BRCA1/BRCA2 DNA repair associated (BRCA1/BRCA2) gene expression in patients with sporadic gastric cancer (GC) who had received postoperative adjuvant chemotherapy. Breast cancer type 1 and 2 susceptibility protein (BRCA1 and BRCA2) expression and BRCA1/BRCA2 mRNA expression were evaluated using immunohistochemistry (IHC) and in-situ hybridization (ISH) on tissue GC microarray tissues, in addition to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results were analyzed for clinicopathological associations. A total of 367 cases of sporadic GC (stages II and III) were subjected to BRCA1 and BRCA2 expression analysis, and for BRCA1 and BRCA2 IHC, 360 cases were informative. A total of 61 cases (16.9%) displayed a loss of BRCA1 and 63 (17.5%) displayed a loss of BRCA2. BRCA1 and BRCA2 ISH results were obtained in 364 cases, of which 98 (26.9%) presented with low expression of BRCA1 mRNA and 148 (40.7%) with low expression of BRCA2 mRNA. In 72 of the 367 cases, BRCA1 and BRCA2 mRNA expression levels were assessed using RT-qPCR, of which 50 (69.4%) and 56 (77.8%) displayed low expression of BRCA1 and BRCA2, respectively. Positive IHC expression of BRCA2 was associated with advanced tumor stage; however, BRCA1 expression was not associated with any clinicopathological parameters. Associations between the RT-qPCR and ISH methods were not significant for either BRCA1 or BRCA2. The results of Kaplan-Meier survival analysis with stage subgrouping revealed no significant differences with regard to survival rate. Of the multivariate analyses, neither BRCA1 nor BRCA2 IHC results were independent prognostic factors. In summary, the present study indicated that BRCA1 and BRCA2, as assessed by IHC, may be used as clinicopathological biomarkers to evaluate the prognosis of sporadic GC.

Proliferation-associated miRNAs-494, -205, -21 and -126 detected by in situ hybridization: expression and prognostic potential in breast carcinoma patients.

To visualize by in situ hybridization (ISH) the levels of a set of proliferation-associated miRNAs and to evaluate their impact and clinical applicability in prognostication of invasive breast carcinoma. Tissue specimen from breast carcinoma patients were investigated for miRNAs-494, -205, -21 and -126. Prognostic associations for levels of miRNAs were analyzed based on complete clinical data and up to 22.5-year follow-up of the patient material (n = 285). For detection of the miRNAs, an automated sensitive protocol applying in situ hybridization was developed. MiRNA-494 indicated prognostic value for patients with invasive breast carcinoma. Among node-negative disease reduced level of miRNA-494 predicted 8.5-fold risk of breast cancer death (p = 0.04). Altered levels and expression patterns of the studied miRNAs were observed in breast carcinomas as compared to benign breast tissue. The present paper reports for the first time on the prognostic value of miRNA-494 in invasive breast cancer. Particularly, detection of miRNA-494 could benefit patients with node-negative breast cancer in identifying subgroups with aggressive disease. Based on our experience, the developed automatic ISH method to visualize altered levels of miRNAs-494, -205, -21 and -126 could be applied to routine pathology diagnostics providing that conditions of tissue treatment, especially fixation delays, are managed.

RNA chromogenic in situ hybridization assay with clinical automated platform is a sensitive method in detecting high-risk human papillomavirus in squamous cell carcinoma

Detection of active human papillomavirus (HPV) is clinically important because its presence has been shown to correlate with favorable clinical outcomes and better response to treatment in oropharyngeal squamous cell carcinomas. Using a clinical automated platform, we compared the performance of commercially available HPV DNA and RNA in situ hybridization (ISH) probes in archival tissues of 57 squamous cell carcinomas. Importantly, a clinical automated platform gives (1) consistent and reproducible results for HPV ISH and (2) better standardization across clinical laboratories. Compared with polymerase chain reaction results, RNA ISH exhibited 93% concordance versus 81% of DNA ISH. RNA ISH was more sensitive than DNA ISH (100% versus 88%) and more specific (87% versus 74%). When only accounting for 2+-3+ positivity, sensitivity was 92% for RNA ISH versus 73% for DNA ISH, highlighting the ease of interpretation. p16 exhibited 96% sensitivity, whereas specificity was only 55%. In 3 cases, both RNA and DNA ISH were positive, whereas polymerase chain reaction results were negative, suggesting that ISH methods might be a more sensitive method. Performing on a clinical automated platform, RNA ISH is sensitive in determining high-risk HPV status in formalin-fixed, paraffin-embedded tissues and has the potential of being a standalone clinical test.

Abstract 361: Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells

Abstract Immunocytochemistry (ICC) utilizes antibodies to visualize the localization of specific proteins in cells while in situ hybridization (ISH) is a useful research tool for examining the presence and changes in levels of gene expression in cells. The ability to simultaneously visualize the co-localization of protein and RNA species in individual cells is necessary to address important biological questions. Additionally, studying both protein and RNA can be useful in cases where expression levels vary between mRNA and protein and to detect post-translational modifications. However, a combined technique has proved challenging due to the incompatibility of ICC and ISH protocols. Furthermore, traditional ISH methods have lacked sensitivity to detect low abundance RNA species and have limited multiplexing capability. Here, we describe a novel technique combining ICC staining with RNA ISH (based on the ViewRNA Assay), which utilizes branched DNA signal amplification technology allowing for single copy mRNA sensitivity. We demonstrate the utility and versatility of this technique using a variety of antibody and probe combinations relevant for cancer research. We compared expression of protein and mRNA of HER2, estrogen receptor alpha, and progesterone receptor in several breast cancer cell lines (MCF7, SKBR3, MDA-MB-175, MDA-MB-231, and BT474) known to express differential levels of these targets. We also compared expression of several microRNAs (let-7a, miR-17, miR-30d, miR-107, and miR-93) known to play a role in cancer development in combination with staining for proteins involved in proliferation (Ki-67), anti-apoptosis (survivin), and cell structure (tubulin). We show successful detection of low abundance RNA species together with various protein markers. This novel technique is a valuable tool not only for cancer researchers, but also for investigators across numerous fields in biological research. Citation Format: Lisa J. Ma, Richard T. Frank, Xiaoxiao Li, Steve Lai, Jeanette Schmidt, Peggy Just, Sara Becker-Catania. Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 361.

Mortality and pathology in birds due to Plasmodium (Giovannolaia) homocircumflexum infection, with emphasis on the exoerythrocytic development of avian malaria parasites.

BackgroundSpecies of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds.MethodsOne individual of Eurasian siskin Carduelis spinus, common crossbill Loxia curvirostra and common starling Sturnus vulgaris were exposed to P. homocircumflexum infection by intramuscular sub-inoculation of infected blood. The birds were maintained in captivity and parasitaemia was monitored until their death due to malaria. Brain, heart, lungs, liver, spleen, kidney, and a piece of breast muscle were examined using histology and chromogenic in situ hybridization (ISH) methods.ResultsAll exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of parenchymal organs.ConclusionPlasmodium homocircumflexum caused death of experimental passerine birds due to marked damage of organs by phanerozoites. Patterns of phanerozoites development and pathology were similar in all exposed birds. Mortality was reported when parasitaemia decreased or even turned into chronic stage, indicating that the light parasitaemia is not always indication of improved health during avian malaria. Application of traditional histological and ISH methods in parallel simplifies investigation of exoerythrocytic development and is recommended in avian malaria research.

In situ DIG-labeling, loop-mediated DNA Amplification (ISDL) for highly sensitive detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is also known as Penaeus stylirostris densovirus (PstDNV). Since it is listed as a reportable crustacean disease by the World Organization for Animal Health (OIE), many countries require testing for IHHNV in shrimp stocks imported for aquaculture, necessitating rapid and highly sensitive detection methods to guarantee and maintain absence of IHHNV in traded stocks. Here we describe a new, highly sensitive method for detection of IHHNV in shrimp tissues. It involves in situ loop-mediated DNA amplification (IS-LAMP) employing digoxigenin-11-dUTP (DIG-dUTP) as part of the deoxynucleotide triphosphate (dNTP) mix. This resulted in DIG-labeling of the DNA amplicons (i.e., in situ, DIG-labeling LAMP or ISDL) allowing direct in situ, immuno-histochemical detection of IHHNV using an anti-DIG antibody conjugated with alkaline phosphatase, and eliminating the lengthy hybridization step of conventional ISH. The target region for the method is at the 3'end of the IHHNV genome that has been reported least likely to give rise to endogenous viral elements (EVE) in the shrimp genome. When compared to the results for IHHN detection by conventional in situ hybridization (ISH) using a DIG-labeled oligonucleotide probe, signals from the ISDL method were stronger and also gave positive results with some tissues unreactive by the conventional ISH method. Additional comparison of the two methods revealed that the proteinase-K digestion step needed for normal ISH was unnecessary for ISDL. This further shortened the assay time and improved the tissue integrity necessary to clearly indicate the location of positive hybridization signals. The ISDL method gave no cross- reactions with tissues of shrimp infected with other common pathogens, and the total time needed to carry out ISDL was one-third of that needed for conventional ISH. Statement of relevanceThis work describes an in situ, DIG-labeling LAMP (ISDL) method, as a new, highly sensitive method for detection of IHHNV in shrimp tissues. The developed method can be used as a confirmatory test carried out with tissue sections to confirm the results from molecular assays, especially when immune-histochemical methods are not available to do so. ISDL is especially important for molecular detection from a new or unexpected location and for molecular detection in newly suspected carriers to determine whether they are actually infected or just mechanical carriers.

Concordance rate between HER2 immunohistochemistry and in situ hybridization in gastric carcinoma: systematic review and meta-analysis.

The aim of this study was to investigate the diagnostic accuracy of HER2 immunohistochemistry (IHC) in gastric carcinoma (GC) through a systematic review, meta-analysis and diagnostic test accuracy review. The current study included 12,679 GC cases and 181 subsets in 45 eligible studies. We performed concordance analysis between HER2 IHC and in situ hybridization (ISH) in GC. Diagnostic test accuracy was analyzed and the area under the curve (AUC) on the summary receiver operating characteristic (SROC) curve was calculated. HER2 amplification rates were 3.0%, 31.8%, and 93.0% in the IHC score 0/1+, 2+, and 3+ groups, respectively. The concordance rates between IHC and ISH were 0.969 (95% confidence interval [CI] 0.962-0.975), 0.393 (95% CI 0.331-0.458) and 0.915 (95% CI 0.882-0.939) in the HER2 IHC score 0/1+, 2+, and 3+ groups, respectively. For all the HER2 IHC score groups, the positive rates were higher in the silver ISH (SISH) subgroup than in the fluorescence ISH (FISH) and chromogenic ISH (CISH) subgroups. In diagnostic test accuracy review, the pooled sensitivity and specificity were 0.86 (95% CI 0.84-0.87) and 0.91 (95% CI 0.90-0.91). The AUC on SROC curve was 0.958. However, there was no significant difference in the values of AUC between the ISH methods. Our results showed that HER2 IHC was well concordant with ISH in HER2 IHC score 0/1+ or 3+. Although this meta-analysis showed higher diagnostic accuracy of HER2 IHC, more detailed criteria for HER2 IHC score 2+ cases will be required to predict HER2 status.

Detection of EBV-encoded Small RNA from Diffuse Large B-cell Lymphoma Patients by RT-PCR Method

Epstein-Barr virus (EBV) has a pathogenic role in several lymphomas including diffuse large B-cell lymphoma (DLBCL). In this study, we detected EBV from formalin-fixed paraffin embedded (FFPE) tissues of DLBCL patients by RT-PCR and compared the sensitivity of the RT-PCR method to in situ hybridization (ISH) method. The RNA was extracted from 91 FFPE samples with DLBCL and amplified with primers targeting EBV-encoded small RNA (EBER) by RT-PCR. When using the RT-PCR method, 13 of 91 patients (14.3%) were positive and among these 13 cases, 7 cases (7.7%) were from > 50-year-old patients that is classified as EBV positive DLBCL of the elderly. In previous results using ISH method, 3 of 91 patients (3.3%) were positive and 2 case (2.4%) were older than 50-year-old. These results indicate that RT-PCR method used in this study shows a higher sensitivity than ISH method. The ratio of male versus female among the EBV positive samples was 1.2:1 with the ratio of male higher. If RT-PCR method having high sensitivity is used simultaneously as well as the ISH method providing the information of the EBV positive cellular location, it is expected that EBV will be more accurately detected.

Ulex Europaeus Agglutinin-1 Is a Reliable Taste Bud Marker for In Situ Hybridization Analyses

Taste signals are received by taste buds. To better understand the taste reception system, expression patterns of taste-related molecules are determined by in situ hybridization (ISH) analyses at the histological level. Nevertheless, even though ISH is essential for determining mRNA expression, few taste bud markers can be applied together with ISH. Ulex europaeus agglutinin-1 (UEA-1) appears to be a reliable murine taste bud marker based on immunohistochemistry (IHC) analyses. However, there is no evidence as to whether UEA-1 can be used for ISH. Thus, the present study evaluated UEA-1 using various histochemical methods, especially ISH. When lectin staining was performed after ISH procedures, UEA-1 clearly labeled taste cellular membranes and distinctly indicated boundaries between taste buds and the surrounding epithelial cells. Additionally, UEA-1 was determined as a taste bud marker not only when used in single-colored ISH but also when employed with double-labeled ISH or during simultaneous detection using IHC and ISH methods. These results suggest that UEA-1 is a useful marker when conducting analyses based on ISH methods. To clarify UEA-1 staining details, multi-fluorescent IHC (together with UEA-1 staining) was examined, resulting in more than 99% of cells being labeled by UEA-1 and overlapping with KCNQ1-expressing cells.

The Clinical Significance of Detecting Bacterial DNA by in situ Hybridization Method in Patients With Decompensated Liver Cirrhosis

Introduction: Patients with decompensated cirrhosis often suffer from various complications such as spontaneous bacterial peritonitis (SBP). However, it is difficult to diagnose SBP or bacteremia because bacteria in ascites or blood cannot be detected accurately by conventional culture method. It has been showed that the method using in situ hybridization (ISH) for detecting the gene of bacteria phagocytized by neutrophil in ascites is possibly useful to diagnose SBP within one day (Enomoto et al. J Hepatology 2012). It is thought that SBP is developed following bacteremia after bacterial translocation from the intestinal tract. Therefore we used the ISH method for blood samples taken from patients with decompensated liver cirrhosis and evaluated the significance of bacterial detection. Methods: Samples of peripheral blood and ascites were collected from 35 patients with ascites caused by liver cirrhosis. Blood samples were examined for bacteria using both conventional blood culture and ISH method. Samples of ascites were also evaluated by conventional culture simultaneously. The ISH method we used was the kit from Fuso Pharmaceuticals (Tokyo, Japan). Results: Eight of 35 patients were diagnosed with SBP. Six of the 8 patients showed positive results using the ISH method while bacteria were detected inonly one case byblood culture and one case by ascites culture. In 27 patients without SBP, 15 patients showed positive results using the ISH method while bacteria were detected in 4 cases by blood culture. The differences between 21 positive and 14 negative results by the ISH method were significant in presence of fever (16 / 5, p < 0.05) and decreased serum albumin levels (2.3 / 2.7, p < 0.05). However, there was no significant difference between administration of antibiotics, C-reaction protein level, and the presence of hepatocellular carcinoma. Notably, no patient who had a positive blood culture had a negative ISH result. Conclusion: The ISH method resulted in a higher positive rate of bacterial detection than culture in patients with decompensated cirrhosis. These results might implicate that “bacterial translocation,” which cannot be proved by conventional culture methods, really occurs. Therefore, it would be better for these patients with positive ISH results to be treated immediately. In patients with decompensated cirrhosis, the ISH method can be helpful for rapid diagnosis and early management of bacteremia and SBP.

Small RNA Detection by in Situ Hybridization Methods.

Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.