Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is ...at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability.
Image-based approaches to single-cell transcriptomics, in which RNA species are identified and counted in situ via imaging, have emerged as a powerful complement to single-cell methods based on RNA ...sequencing of dissociated cells. These image-based approaches naturally preserve the native spatial context of RNAs within a cell and the organization of cells within tissue, which are important for addressing many biological questions. However, the throughput of these image-based approaches is relatively low. Here we report advances that lead to a drastic increase in the measurement throughput of multiplexed error-robust fluorescence in situ hybridization (MERFISH), an image-based approach to single-cell transcriptomics. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule fluorescence in situ hybridization (smFISH) to read out these barcodes. Here we increase the throughput of MERFISH by two orders of magnitude through a combination of improvements, including using chemical cleavage instead of photobleaching to remove fluorescent signals between consecutive rounds of smFISH imaging, increasing the imaging field of view, and using multicolor imaging. With these improvements, we performed RNA profiling in more than 100,000 human cells, with as many as 40,000 cells measured in a single 18-h measurement. This throughput should substantially extend the range of biological questions that can be addressed by MERFISH.
Supernumerary B chromosomes (Bs) are genomic parasitic components, originating from the A complement via chromosomal rearrangements, which follow their own evolutionary trajectories. They often ...contain repetitive DNAs, some shared with regular chromosomes and some newly evolved. Genomic composition, origin and evolution of Bs have been analysed in the chromosomally variable Prospero autumnale complex. Two rDNAs and a satellite DNA (PaB6) from regular chromosomes were mapped to Bs of 26 plants from three diploid cytotypes, their hybrids and polyploid derivatives. In homoploid diploid hybrids, genomic in situ hybridization (GISH) allowed B painting with the parental DNAs. Bs were structurally variable and highly enriched in 5S rDNA and satDNA PaB6, and rarely in 35S rDNA. Eleven combinations of rDNA and PaB6 localization were observed. The quantities of PaB6 in Bs and regular chromosomes were not correlated, suggesting amplification mechanisms other than recombination. PaB6 and 5S rDNA amounts increased with increasing ploidy level. GISH revealed two independent origins of Bs. The structural variation, repeat content, repeat‐type fluctuations and differing genomic affinities of Bs in different cytotypes suggest that they represent young proto‐B chromosomes. Bs in P. autumnale probably form recurrently as by‐products of the extensive genome restructuring within this chromosomally variable species complex.
García‐García E, Gómez‐Martín C, Angulo B, Conde E, Suárez‐Gauthier A, Adrados M, Perna C, Rodríguez‐Peralto J L, Hidalgo M & López‐Ríos F (2011) Histopathology59, 8–17
Hybridization for human ...epidermal growth factor receptor 2 testing in gastric carcinoma: a comparison of fluorescence in‐situ hybridization with a novel fully automated dual‐colour silver in‐situ hybridization method
Aims: Amplification of the human epidermal growth factor receptor 2 (HER2) gene has been reported in gastric carcinoma (GC). Accordingly, trastuzumab plus chemotherapy has recently become the new standard treatment for HER2‐positive advanced GCs. The aim was to compare the alleged gold standard for hybridization fluorescence in‐situ hybridization (FISH) with a novel, fully automated brightfield dual‐colour silver‐enhanced in‐situ hybridization (SISH) method.
Methods and results: The studies series was comprised of 166 GC samples. Additionally, tumours with discordant results obtained by FISH and SISH were analysed by real‐time quantitative polymerase chain reaction (PCR) with the LightMix kit HER‐2/neu. Of the samples, 17.5% and 21% were amplified by FISH and SISH, respectively. Heterogeneity was identified in up to 52% of cases. In 96.4% of cases, FISH showed the same results as SISH. All six discordant cases were positive by SISH and negative by FISH. On review of the FISH slides, all contradictory cases were polysomic and were confirmed to be negative for amplification by real‐time PCR. Interestingly, all ratios in this latter group were between 2.06 and 2.50, so setting the cut‐off for amplification at ≥3 resulted in perfect concordance.
Conclusions: Dual‐colour SISH represents a novel method for the determination of HER2 status in GC.
Aims
The wide variety of affected organ systems associated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection highlights the need for tissue‐specific evaluation. We compared ...placentas from SARS‐CoV‐2‐positive and SARS‐CoV‐2‐negative women in our hospital in New York City, which became the epicenter of the coronavirus disease 2019 pandemic in March 2020. To date, some limited studies have been published on placentas from SARS‐CoV‐2‐positive women. The aim of our study, in addition to describing histomorphology, was to utilize in‐situ hybridization (ISH) for the S‐gene encoding the spike protein and immunohistochemistry (IHC) with the monoclonal SARS‐CoV‐2 spike antibody 1A9 for placental evaluation.
Methods and results
In this study, 51 singleton, third‐trimester placentas from SARS‐CoV‐2‐positive women and 25 singleton, third‐trimester placentas from SARS‐CoV‐2‐negative women were examined histomorphologically according to the Amsterdam Criteria and with ISH and/or IHC. The corresponding clinical findings and neonatal outcomes also were recorded. Although no specific histomorphologic changes related to SARS‐CoV‐2 were noted in the placentas, evidence of maternal–fetal vascular malperfusion was identified, with placentas from SARS‐CoV‐2‐positive women being significantly more likely to show villous agglutination (P = 0.003) and subchorionic thrombi (P = 0.026) than placentas from SARS‐CoV‐2‐negative women. No evidence of direct viral involvement was identified with ISH and IHC.
Conclusions
In this study, third‐trimester placentas from SARS‐CoV‐2‐positive women were more likely to show evidence of maternal–fetal vascular malperfusion; however, ISH and IHC provided no evidence of direct viral involvement or vertical transmission.
Aims
Pleomorphic adenoma gene 1 (PLAG1) gene rearrangement is the most common genetic abnormality in pleomorphic adenoma (PA), resulting in overexpression of PLAG1 protein. PA and carcinoma ex ...pleomorphic adenoma (CA ex‐PA) can mimic various benign and malignant salivary gland tumours. The aims of this study are to evaluate the sensitivity and specificity of PLAG1 immunohistochemistry (IHC) in the differential diagnosis of PA and CA ex‐PA and to compare the PLAG1 immunohistochemical results to PLAG1 gene abnormalities as detected by fluorescence in‐situ hybridisation (FISH).
Methods and results
PLAG1 immunostaining was performed on 83 salivary gland tumours, including 23 PA, 15 CA ex‐PA and 45 other salivary gland tumours. In addition, PLAG1 FISH was performed in 44 cases for the presence of gene rearrangements/amplifications. The results showed high sensitivity of PLAG1 IHC in 96% of PA; however, discordant results between PLAG1 FISH abnormalities and IHC were noted in 15 of 44 cases (34%). Seven PA, four de‐novo myoepithelial carcinomas and one basal cell adenocarcinoma had negative FISH results, but were positive for IHC; while three salivary duct carcinomas (SDC) ex‐PA were positive for FISH but negative for IHC. PLAG1 IHC can differentiate CA ex‐PA from de‐novo SDC (P = 0.02), but not from de‐novo myoepithelial carcinoma. PLAG1 IHC is a sensitive marker for PA. This could be due to PLAG1 gene abnormalities beyond FISH resolution.
Conclusions
A negative PLAG1 IHC might be helpful in excluding a PA diagnosis. Interestingly, in the context of CA ex‐PA, FISH is more sensitive than IHC in detecting PLAG1 abnormalities.
The freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount ...fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge.
We show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts.
The modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms.
Aims
Approximately 60–70% of high‐grade round‐cell sarcomas that lack the Ewing sarcoma breakpoint region 1 (EWSR1) rearrangement harbour a rearrangement of the CIC gene, most commonly CIC–DUX4. ...Recent studies have established that CIC‐rearranged sarcomas constitute a distinct group characterized by recognizable histology and immunoprofiles, such as positivity for ETV4 and WT1 and negativity for NKX2.2. Although these sarcomas are diagnosed increasingly in practice by fluorescence in‐situ hybridization (FISH) with CIC break‐apart probes, the optimal modality to diagnose these sarcomas has not been determined. In this study, we describe four round‐cell sarcomas that showed false‐negative results by CIC break‐apart FISH assays.
Methods and results
These sarcomas showed characteristic histology of CIC‐rearranged sarcomas, and all were immunohistochemically positive for ETV4 and WT1 and negative for NKX2.2. Although FISH showed non‐atypical negative signals for CIC rearrangement, high‐throughput RNA sequencing identified CIC–DUX4 and its fusion breakpoint in all cases. Their clinical and histological findings, as well as fusion points determined by RNA sequencing, did not differ significantly from those of nine FISH‐positive CIC–DUX4 sarcoma cases. We estimated that the FISH false‐negative rate for CIC‐rearranged sarcomas was 14%. Although neither histology nor immunoprofiles (e.g. ETV4 and WT1) are entirely sensitive or specific for CIC‐rearranged sarcomas, the observation that these four cases were identified successfully by such phenotypes suggested their practical utility.
Conclusions
CIC break‐apart FISH assays missed a significant minority of CIC–DUX4 sarcomas, and full awareness of typical morphology and judicious immunohistochemical work‐ups, including analyses of ETV4 and WT1, should complement diagnostic assessment.
Fluorescence in situ hybridization (FISH) was developed more than 30 years ago and has been the most paradigm-changing technique in cytogenetic research. FISH has been used to answer questions ...related to structure, mutation, and evolution of not only individual chromosomes but also entire genomes. FISH has served as an important tool for chromosome identification in many plant species. This review intends to summarize and discuss key technical development and applications of FISH in plants since 2006. The most significant recent advance of FISH is the development and application of probes based on synthetic oligonucleotides (oligos). Oligos specific to a repetitive DNA sequence, to a specific chromosomal region, or to an entire chromosome can be computationally identified, synthesized in parallel, and fluorescently labeled. Oligo probes designed from conserved DNA sequences from one species can be used among genetically related species, allowing comparative cytogenetic mapping of these species. The advances with synthetic oligo probes will significantly expand the applications of FISH especially in non-model plant species. Recent achievements and future applications of FISH and oligo-FISH are discussed.
Right detection of anaplastic lymphoma kinase (ALK) gene rearrangement is pivotal to selection of patients with lung adenocarcinoma for ALK-targeted therapy. We explored the potential of combination ...of immunohistochemistry (IHC) screening and fluorescence in situ hybridization (FISH) as an affordable practice. We analyzed 410 unselected lung adenocarcinomas by ALK IHC (D5F3 clone) and FISH. Some equivocal cases were further analyzed by RT-PCR. The EGFR mutation was detected by pyrosequencing assay. In total 368 cases which got all IHC, FISH, EGFR mutation results were eligible for analysis. Cases were evaluated as IHC score 3+ (n = 26), score 2+ (n = 9), score 1+ (n = 51), and score 0 (n = 282), respectively. 23 of 26 IHC 3+ and 5 of 9 IHC 2+ cases were FISH positive, whereas 3 of 26 IHC 3+, 4 of 9 IHC 2+ and all 333 IHC 1+/0 cases were FISH negative. If considering FISH as the standard, the sensitivity and specificity of ALK IHC 3+/2+ as ALK positive were 100% and 97.9%, respectively. Three IHC 3+ cases reported as FISH "negative" were actually ALK positive confirmed by ALK RT-PCR or re-detected. Based on the final classify, ALK IHC 3+/2+ was 100% sensitive and 98.8% specific. However, FISH was 90.3% sensitive and 100% specific. IHC 2+ was regarded as equivocal and need to be confirmed by FISH or RT-PCR. In the 368 cases, 8.4% cases had ALK positive, 52.2% cases had EGFR mutation, and only one case had a coexisting. Manually semiquantitative ALK IHC (primary antibody D5F3 coupled with secondary DAKO Envision system) used as the initial screening combined with auxiliary FISH confirmation is a reliable, economical approach to identify ALK positive lung adenocarcinoma. The IHC can find some ALK positive cases which would be missed by FISH only.
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