Programmable in situ amplification for multiplexed imaging of mRNA expression

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization

Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

<p>Abstract</p> <p>Background</p> <p><it>HER-2 </it>gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence <it>in situ </it>hybridization (FISH). These procedures permit correlation between <it>HER-2 </it>expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic <it>in situ </it>hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas.</p> <p>Methods</p> <p>To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH.</p> <p>Results</p> <p>The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of <it>HER-2 </it>status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and <it>HER-2 </it>transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and <it>HER-2 </it>downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between <it>HER-2 </it>downexpression and the involvement of less than four lymph nodes (<it>P </it>= 0.0350).</p> <p>Conclusion</p> <p>Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the <it>HER-2 </it>gene.</p

Historia de la Facultad de Ciencias de la Educación de Paraná (Argentina), 1973-1983

En este artículo se procura reconstruir la historia de la Facultad de Ciencias de la Educación de la Universidad Nacional de Entre Ríos durante un lapso acotado a una década del S. XX. A partir de las múltiples variables que componen la realidad universitaria y su contexto y con la pretensión de lograr la comprensión de la vida institucional, se enhebra el análisis de los proyectos institucionales, las orientaciones ideológico-académicas, los perfiles formativos en pugna, el accionar de los sujetos, la inserción de la facultad en el medio, los conflictos con ciertas tradiciones a partir de vertebrar un importante y heterogéneo corpus documental (fuentes oficiales, archivos institucionales, planes de estudio, programas de cátedra, información periodística, entrevistas a informantes calificados y padrón de egresados)

Validation of the 4B5 rabbit monoclonal antibody in determining Her2/neu status in breast cancer

HER2 overexpression in breast cancer is associated with worse clinical outcome. To select patients for anti-Her2-based therapy immunohistochemistry is commonly performed as a first step to assess Her2 status. However, interobserver and interlaboratory variability can significantly compromise adequate assessment of Her2 status. In addition, immunohistochemistry does not always result in an unambiguous test result requiring additional testing for Her2 gene amplification. This study aimed to improve the reliability of Her2 immunohistochemistry by using rabbit monoclonal antibody 4B5 as an alternative to mouse monoclonal antibody CB11 routinely used in our laboratory. Therefore, 283 breast adenocarcinomas were included in a tissue microarray. Immunohistochemistry using the 4B5 and CB11 antibodies, and fluorescence and chromogenic in situ hybridization (FISH or CISH) were performed. Immunohistochemistry was scored by two independent investigators. We found that 4B5 staining was more distinct than CB11 staining. For CB11 staining, there were 12% (BV) and 5% (JW) 2 + scores compared with 4% (BV) and 2% (JW) for 4B5. There was a strong trend towards higher interobserver agreement for 4B5 compared with CB11 (4B5: kappa 0.87, 95% CI 0.79-0.96; CB11: kappa 0.77, 95% CI 0.66-0.88). There were no significant differences in sensitivity, specificity and predictive values between CB11 and 4B5. Our results indicate that the 4B5 antibody provides more robust assessment of immunohistochemical Her2/neu status and will reduce the number of gene amplification tests compared with CB11. However, for tumours with a 2 + score additional gene amplification measurement using FISH or CISH remains necessary. Modern Pathology (2009) 22, 879-886; doi: 10.1038/modpathol.2009.37; published online 20 March 2009