Plate in situ hybridization (PISH) as a time and cost effective RNA expression assay to study phenotypic heterogeneity in a population of cultured murine cells at single cell resolution.

OBJECTIVES: Regenerative medicine approaches using reprogrammed or transdifferentiated cells require efficient single cell expression profiling to analyze culture homogeneity for quality control and recipients' safety. RESULTS: While antigen-antibody based systems have been developed for several proteins, probing at the mRNA level allows for more flexibility, faster adaption to the ever increasing new data from next generation sequencing and increased specificity, especially for genes of conserved gene families. CONCLUSIONS: We developed a time and cost effective expression profiling assay for monolayer cell culture in 96-well plates based on RNA in situ hybridization, termed PISH, at single cell resolution.

Chromogen detection of microRNA in frozen clinical tissue samples using LNA™ probe technology.

Specific chromogen- and fluorescence-based detection of microRNA by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded (FFPE) tissue sections has been facilitated by locked nucleic acid (LNA)-based probe technology and can be performed within a single working day. In the current method paper, we present a similar simple 1-day ISH method developed for cryostat sections obtained from clinical cryo-embedded tissue samples. The presented chromogen-based ISH method does not involve proteolytic pretreatment, which is mandatory for FFPE sections, but still retains a sensitivity level similar to that obtained in FFPE sections. The LNA-based ISH method is not only applicable in situations where only access to cryo-embedded material is possible, but it also has a potential use if combining microRNA ISH with immunohistochemistry in double fluorescence staining with antibodies not being compatible with proteolytic predigestion.

Zinc-based fixation for high-sensitivity in situ hybridization: a nonradioactive colorimetric method for the detection of rare transcripts on tissue sections.

Nonradioactive colorimetric in situ hybridization (NoRISH) has been widely applied to analyze gene expression at the single-cell level. Zinc fixation is time efficient and provides excellent tissue morphology. Furthermore, it improves the preservation of the RNA, facilitating the detection of rare transcripts or the identification of expressing cells scattered within a tissue. Here we present a rapid, highly sensitive NoRISH method that uses a zinc-salt-based fixative and is especially suitable for the study of genes expressed at low levels and/or in a small number of cells within a structure.

Detection of small noncoding RNAs by in situ hybridization using probes of 2'-O-methyl RNA + LNA.

In situ hybridization is a powerful method to provide information about contextual distribution and cellular origin of nucleic acids, e.g., in formalin-fixed paraffin-embedded (FFPE) samples of tissue. Particularly the recently discovered classes of noncoding RNA (ncRNA) including endo-siRNAs and microRNAs require such a technique to enable their study and visualization in natural contexts, and in the last decade, many advances have been made, increasing our ability to specifically detect small ncRNAs. One of the key developments has been the demonstration of the superiority of using locked nucleic acid (LNA)-modified DNA probes for the detection of ncRNA in tissue. Here, we describe an alternative in situ hybridization protocol employing oligonucleotide probes consisting of combinations of LNA and 2´-O-methyl RNAs that under optimized hybridization buffer conditions can provide a highly sensitive assay performance with only 1 h hybridization time.

Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.

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.

Limited human epidermal growth factor receptor 2 discordance in metastatic breast cancer patients treated with trastuzumab, a population based study.

BACKGROUND: Accurate assessment of the human epidermal growth factor receptor 2 (HER2) in breast cancer is essential for proper treatment decisions. HER2 positivity confirmation rates in breast cancer trials by central testing pathology laboratories were reported to be approximately 85%. The aim of our study was to assess in a population based sample concordance of HER2 status in metastatic breast cancer (MBC) patients locally tested HER2 positive and treated with trastuzumab. Moreover cost-effectiveness of in situ hybridisation (ISH) in patients with an immunohistochemical score 3+ (IHC3+) was explored. METHODS: MBC patients treated between 2005 and 2009 with trastuzumab-based therapy in North East Netherlands were identified by a survey of hospital pharmacies. Primary tumour samples were retested centrally for HER2 status using 1 immunohistochemical (IHC) method and two methods using ISH on tissue micro-arrays. Potential discordant patients were retested on whole tumour slides. HER2 positivity was defined as: (1) ISH amplification (according to American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) clinical practice Guideline Update) and (2) when ISH failed an IHC score of 3+. Cost-effectiveness was estimated using potential ISH and treatment costs. RESULTS: HER2 status could be retested in 174 of 194 (90%) patients. The HER2 concordance rate was 87%. The 21 discordant patients were in the 67% due to primary HER2 testing with only IHC. Overall survival of HER2 discordant and concordant patients was not significantly different (18 versus 25months, p=0.131). Structural ISH in the case of IHC3+ has an estimated potential saving of €87,710 per 100 patients. CONCLUSION: HER2 concordance in a population based study is comparable to those described in selected populations. Discordance is mostly due to testing with only IHC. ISH in the case of IHC3+ is cost-effective.

Chromogenic in situ hybridisation (CISH) is a powerful method to detect ALK-positive non-small cell lung carcinomas.

AIM: We assessed the potential of a chromogenic in situ hybridisation (CISH) assay in comparison with quantitative reverse transcription (RT)-PCR (qPCR) to detect anaplastic lymphoma kinase (ALK) break apart-positive lung carcinomas. METHODS: Dual-colour CISH using a break apart probe for the ALK gene on 2p23 was performed with 181 formalin-fixed, paraffin-embedded tissue and agar block sections from 175 cases of non-small cell lung carcinomas (NSCLC). Stained slides were analysed with a standard bright-field microscope at 1000× magnification by counting signals from 60 non-overlapping nuclei from three different tumour areas. Samples with ≥15% of positive nuclei were judged as ALK break apart-positive. All samples were simultaneously analysed by qPCR for EML4-ALK to validate CISH results, and positive samples were subject to Sanger sequencing. RESULTS: CISH was successful with 173 of 181 hybridised samples (96%), and seven ALK break apart-positive cases were detected. CISH signals were specific and distinct for both colours. All positive cases were confirmed by qPCR and Sanger sequencing, and concordance between CISH and qPCR was 100%. Nearly all samples (9/10) which failed by qPCR were accessible to CISH analysis. CONCLUSIONS: CISH is a very reliable, convenient and inexpensive method to detect ALK-positive NSCLC. CISH success rate is comparably high as with qPCR, and it detects all ALK break apart events in a single assay. It is of special value when RNA quality is poor, or when small biopsies with a very limited amount of tumour cells have to be analysed.

An economical and practical method for whole-mount in situ hybridization for mouse embryos and organs.

Whole-mount in situ hybridization (WISH) is a useful method for detecting specific gene expression patterns at their site of action during embryonic development. Traditional WISH methods are costly and suitable only for mouse embryos younger than 11.5 days. We present here an economical and practical in situ hybridization method using DIG-labeled RNA probes. We changed the conditions in several steps to make the WISH method suitable for whole mouse embryos from embryonic days 9.5 to 12.5 and for older stage mouse embryonic organs. We performed all steps in one microcentrifuge tube up to the staining steps to avoid losing or damaging the mouse embryos. We re-used the solutions and materials to make the method more economical and suitable for less sophisticated laboratories. We also performed ß-galactosidase staining on Tb × 18 Cre/Rosa26/LacZ mouse embryos; the results agreed with the in situ hybridization results. Finally, we sectioned the specimens after hybridization and ß-galactosidase staining; the results agreed with the literature.

HER 2 status in invasive breast cancer: immunohistochemistry, fluorescence in-situ hybridization and chromogenic in-situ hybridization.

INTRODUCTION: HER2/neu gene status in breast cancers can be evaluated by targeting protein and gene - immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH). Recent studies have shown chromogenic in-situ hybridization (CISH) as a relatively cheaper alternative. MATERIALS AND METHODS: Forty-three nonconsecutive, randomly selected primary invasive breast cancer cases were evaluated for c-erbB-2 (HER2 protein) by IHC and gene amplification by FISH and CISH. Results of each of the same were compared. RESULTS: CISH showed approximately 90% and 100% concordance for IHC negative and positive cases, respectively; while approximately 94.4% and 91% concordance with FISH amplified and non-amplified cases, respectively. CONCLUSION: This study showed feasibility of incorporation of CISH as a low cost option in routine management of breast carcinoma in the Indian setting. Secondly, reconfirmation of IHC negative and positive cases can be done by CISH.

Epstein-Barr virus (EBV)-encoded RNA: automated in-situ hybridization (ISH) compared with manual ISH and immunohistochemistry for detection of EBV in pediatric lymphoproliferative disorders.

Detection of Epstein-Barr virus (EBV) may be achieved by various methods, including EBV-encoded RNA (EBER) in-situ hybridization (ISH) and immunohistochemistry (IHC) for latent membrane protein (LMP-1). We compared novel automated ISH and IHC techniques in pediatric lymphoproliferative disorders with results obtained by manual ISH. Thirty-seven pediatric cases previously studied by manual EBER ISH (including 18 EBER-positive, 15 EBER-negative, and 4 EBER-equivocal cases) were used for the study. Automated EBER ISH and automated LMP-1 IHC were performed using the BondMax autostainer and prediluted EBER probe and EBV cell surface 1 to 4 at 1:50 dilution, respectively. Results of each of the automated techniques for EBV detection were compared with results by manual EBER ISH. Compared with manual EBER ISH as the gold standard, automated ISH had a sensitivity and specificity of 94% and 69%, respectively, accuracy of 83%, positive predictive value (PPV) of 79%, and negative predictive value (NPV) of 90%. Automated IHC had a sensitivity of 44%, specificity of 93%, accuracy of 67%, PPV of 88%, and NPV of 59%. Automated ISH and IHC correlated significantly (P < 0.045). Automated ISH is useful for diagnosis of EBV-related pediatric neoplasms, being easy to perform and interpret and requiring only the technologist's time to set up and having a high sensitivity and NPV The automated IHC protocol is of too low sensitivity for routine use, although results show high specificity and PPV.

Localization of HSP single-copy genes by inexpensive, permanent non-fluorescent in situ hybridization on meiotic chromosomes of the grasshopper Schistocerca pallens (Acrididae).

There have been many studies on Schistocerca gregaria and Locusta migratoria, which are important grasshopper pests in many parts of the world. However, the main pest grasshopper species in Brazil, S. pallens, Rhammatocerus schistocercoides and Stiphra robusta, are very poorly characterized genetically. We adapted a permanent in situ hybridization method to extend the genetic characterization of S. pallens by mapping the single-copy genes Hsp70, Hsp83, Hsp27, and Ubi on meiotic chromosomes. Hsp70 was mapped on the L2 chromosome, in which 82% of the signals were observed. Hsp83 was mapped on a medium-sized chromosome, on which 81% of the signals were observed, tentatively identified as M7. The hybridization signals for the Hsp27 gene were detected on the L1 chromosome at a frequency of 58%. The main hybridization site of the Ubi probe was on the L2 chromosome, with 73% of the signals. All mapped genes also presented secondary hybridization signals, always at frequencies below 30%. These are the first single-copy genes mapped for S. pallens and also for the Acrididae family. Since the Acrididae generally present very similar karyotypes, these data are useful as new landmarks for chromosome identification and as a tool for phylogenetic studies on the genus Schistocerca and for comparison with other insects.

Localization of HSP single-copy genes by inexpensive, permanent non-fluorescent in situ hybridization on meiotic chromosomes of the grasshopper Schistocerca pallens (Acrididae)

There have been many studies on Schistocerca gregaria and Locusta migratoria, which are important grasshopper pests in many parts of the world. However, the main pest grasshopper species in Brazil, S. pallens, Rhammatocerus schistocercoides and Stiphra robusta, are very poorly characterized genetically. We adapted a permanent in situ hybridization method to extend the genetic characterization of S. pallens by mapping the single-copy genes Hsp70, Hsp83, Hsp27, and Ubi on meiotic chromosomes. Hsp70 was mapped on the L2 chromosome, in which 82% of the signals were observed. Hsp83 was mapped on a medium-sized chromosome, on which 81% of the signals were observed, tentatively identified as M7. The hybridization signals for the Hsp27 gene were detected on the L1 chromosome at a frequency of 58%. The main hybridization site of the Ubi probe was on the L2 chromosome, with 73% of the signals. All mapped genes also presented secondary hybridization signals, always at frequencies below 30%. These are the first single-copy genes mapped for S. pallens and also for the Acrididae family. Since the Acrididae generally present very similar karyotypes, these data are useful as new landmarks for chromosome identification and as a tool for phylogenetic studies on the genus Schistocerca and for comparison with other insects.

HPV DNA testing in the triage of atypical squamous cells of undetermined significance (ASCUS): cost comparison of two methods.

Human papillomavirus (HPV) DNA testing for triage of cervical cytologies showing atypical squamous cells of undetermined significance (ASCUS) has become the standard of practice. Currently, Hybrid Capture II (HCII) is the preferred method for ASCUS triage. In situ hybridization for HPV represents an alternative to HCII and appears to have a superior specificity but is more expensive. We compare the reimbursement rates of ASCUS triage (HPV high risk) using the methods of HCII and INFORM (in situ hybridization for HPV) in a series of 431 ASCUS patients. The patients were followed for 1 yr, during which each patient had either colposcopic biopsy or follow-up cervical cytology after ASCUS HPV DNA triage. Eighty-nine patients were excluded from the analysis because of incomplete follow-up. The HPV triage percentages, colposcopic biopsy positivity rates and cervical cytology positivity percentages were calculated for each method. The reimbursement rates of the tests/procedures used in the analysis were those in effect at the University of Utah in 2003. The total triage and follow-up reimbursement costs were calculated for HCII and INFORM and compared.HCII referred 19.9% of patients to colposcopy, with a biopsy positivity rate of 25.6% for dysplasia. INFORM referred 11.8% of patients to colposcopy, of whom 34% had a biopsy diagnosis of dysplasia. HCII negative cases revealed 19% to have ASCUS or higher on the follow-up cervical cytology, while 19.9% of INFORM negative cases had a reading of ASCUS or higher at follow-up cytologic examination. The 1-yr HPV DNA triage and follow-up reimbursements for HCII were 316,942.00 US dollars per 1,000 women, and for the INFORM methodology, the reimbursements were 369,484.00 US dollars per 1,000 women. The INFORM method was associated with higher specificity and sent fewer (41%) patients to colposcopy than did HCII. Although this smaller referral rate reduced reimbursement costs associated with colposcopy, the increased reimbursement paid for follow-up cytologies and office visits of HPV DNA negative patient and the greater cost of the INFORM test results in higher overall reimbursement for INFORM. Based on these costs and diagnostic accuracies, it appears that the INFORM HPV technology represents a viable option to HCII ASCUS triage. INFORM HPV appears to be 16% more expensive than HCII but has the advantage of sending 41% fewer women to colposcopy.

Chemiluminescent detection of genetic polymorphisms based on mismatch hybridization: application to cytochrome P4501A1.

An assay that makes use of differences in thermal stability between perfectly and imperfectly matched hybrids in combination with a sensitive chemiluminescence detection system was developed and applied to the identification of CYP1A1 polymorphisms. In this assay, two oligonucleotide probes for each polymorphic site were designed: one perfectly matching the wild type allele, the other perfectly matching the mutant allele. The genotypes were determined by calculating the ratio of signals obtained from the two probes. The method described here allows for the rapid, simple and cost-effective detection of DNA polymorphisms. Compared with fluorescence- and microarray-based assays, this method provides an alternative for genotyping where costly equipment or specialized reagents are not available.

In situ detection of a PCR-synthesized human pancentromeric DNA hybridization probe by color pigment immunostaining: application for dicentric assay automation.

We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.

Methods and reagents. Degraded DNA and gel tornados.

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses a case of inexplicable DNA degradation and tornados seen in agarose gels. For details on how to partake in the newsgroup, see the accompanying box.

Non-isotopic in situ hybridization of human papilloma virus on histologic sections: an amended protocol.

The authors report on their experience with an HPV non-radioactive in situ hybridization kit and describe the favorable results gained with the amended protocol, which are as follows: 1. The application of a decreased amount of both the probe and the chromogen substrate did not alter the quality of reactions. Therefore we were able to make 60 reactions instead of the originally suggested 21. 2. The proteolytic enzyme digestion time could be prolonged by changing proteinase-K for pepsin which intensifies the signal of hybridization. 3. By changing the order of hybrid detection and posthybridization washing, we succeeded in removing the excess amount of probe-ABC-AP-BAAV-ABC-AP conglomerates without losing the target sequence. 4. Using alkaline phosphatase or ABC-AP-BAAV-ABC-AP complex instead of peroxidase it was possible to demonstrate a very low number of gene copies, even if they were not detectable following the original instructions.

Detection of cytomegalovirus by in situ hybridization using a digoxigenin-tailed oligonucleotide.

A non-isotopic in situ hybridization procedure was used to detect cytomegalovirus (CMV) sequences within routinely fixed tissue. A digoxigenin-tailed oligonucleotide was hybridized to sections of specimens obtained at autopsy from 2 patients with CMV infection. Hybrids were revealed by an alkaline phosphatase-conjugated anti-digoxigenin antibody. Serial sections were also assayed for the presence of CMV by in situ hybridization with a biotin-labelled cDNA probe and by immunohistochemistry and routinely stained for morphological evaluation. Results show that the two in situ hybridization procedures are equally sensitive but superior to the immunohistochemical detection of the viral antigen. Most cells positive for CMV DNA had the cytopathological features characteristic of CMV infection. A minor population of infected cells lacking morphological changes was also found. We recommend the routine application of the oligonucleotide-based assay because it is specific, easy and less expensive than other similar procedures.