Molecular profiling of multiplexed gene markers to assess viability of ex vivo human colon explant cultures

© Janice E. Drew et al. 2015; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Acknowledgments The authors would like to thank the patients who kindly donated tissue samples, Sally Chalmers of the Tayside Tissue Bank for her help with collecting of the tissue donor samples, Emma Moss for advice on human colon dissection and explant culture, and Claus Dieter Mayer, Biomathematics and Statistics Scotland, for advice on statistical analysis. This work was supported by the Scottish Government (GT403), Scottish Universities Life Science Alliance, and TENOVUS Scotland.Peer reviewedPublisher PD

Predictive gene signatures:molecular markers distinguishing colon adenomatous polyp and carcinoma

Funding: This study was supported by the Scottish Government's Rural and Environment Science and Analytical Services Division Food, Land and People Programme GT403 (http://www.scotland.gov.uk/Topics/Research/About/EBAR/StrategicResearch/future-research-strategy/Themes), Scottish Universities Life Science Alliance Translational Biology Studentship 10/09, (http://www.sulsa.ac.uk/), NHS Grampian Endowment Fund 12/07 (http://www.nhsgrampian.co.uk/nhsgrampian/gra_display_hospital.jsp?pContentID=65&p_applic=CCC&p_service=Content.show&). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.Peer reviewedPublisher PD

Enzymatic on-Chip Enhancement for High Resolution Genotyping DNA Microarrays

Antibiotic resistance among pathogenic microorganisms is emerging as a major human healthcare concern. While there are a variety of resistance mechanisms, many can be related to single nucleotide polymorphisms and for which DNA microarrays have been widely deployed in bacterial genotyping. However, genotyping by means of allele-specific hybridization can suffer from the drawback that oligonucleotide probes with different nucleotide composition have varying thermodynamic parameters. This results in unpredictable hybridization behavior of mismatch probes. Consequently, the degree of discrimination between perfect match and mismatch probes is insufficient in some cases. We report here an on-chip enzymatic procedure to improve this discrimination in which false-positive hybrids are selectively digested. We find that the application of CEL1 Surveyor nuclease, a mismatch-specific endonuclease, significantly enhances the discrimination fidelity, as demonstrated here on a microarray for the identification of variants of carbapenem resistant <i>Klebsiella pneumoniae</i> carbapenemases and monitored by end point detection of fluorescence intensity. Further fundamental investigations applying total internal reflection fluorescence detection for kinetic real-time measurements confirmed the enzymatic enhancement for SNP discrimination

<i>In situ</i> hybridisation of <i>LGR5</i> transcripts in human colon (A) normal, (B) adenomatous polyp and (C) carcinoma.

<p>Emulsion autoradiographs showing expression of <i>LGR5</i> in discrete single cells (arrow) in epithelium (ep) in normal (A) and extensive expression in epithelium (ep) of adenomatous polyp (B) and carcinoma (C) in bright field and corresponding adjacent dark field images. Antisense hybridised tissue sections are shown to the left with sense hybridised tissue sections adjacent to the right (n = 5). Bar = 20 µm.</p

Relative gene expression levels in human colon normal, adenomatous polyp and carcinoma tissue generated using the GeXP hCellMarkerPlex assay.

<p>Gene expression is normalised to internal reference gene <i>UBE2D2</i> in the hCellMarkerPlex. The letters indicate significant (p<0.05) difference in gene expression between ‘a’ normal (n = 24) and either adenomatous polyp (n = 17) or carcinoma (n = 19), ‘b’ normal, adenomatous polyp and carcinoma, ‘c’ normal and carcinoma, ‘d’ normal and adenomatous polyp and ‘e’ carcinoma and either normal of adenomatous polyp.</p

Gene expression of (A) long and (B) short form variants of <i>MS4A12</i> in human colon normal, adenomatous polyp and carcinoma tissue.

<p>Gene expression is normalised to reference gene <i>UBE2D2</i>. The asterisk (*) indicates significant decrease in expression levels of adenomatous polyp compared to normal, p<0.005. (C) – (D). <i>In situ</i> hybridisation of <i>MS4A12</i> transcripts in human colon (C) normal, (D) adenomatous polyp and (E) carcinoma. Emulsion autoradiographs showing expression of <i>MS4A12</i> at luminal epithelial surface (ep) of normal (C) in bright field and corresponding dark field images in antisense (left) and sense (right) hybridised tissue sections (n = 5). <i>MS4A12</i> is largely absent in adenomatous polyp (D) and localised in discrete areas of epithelium in carcinoma (E). Bar = 20 µm.</p

<i>In situ</i> hybridisation of <i>NOX1</i> transcripts in human colon (A) normal, (B) adenomatous polyp and (C) carcinoma.

<p>Emulsion autoradiographs showing expression of <i>NOX1</i> in epithelium (ep) in bright field and corresponding adjacent dark field images in antisense (left) and sense (right) hybridised tissue sections (n = 5). Bar = 20 µm.</p

Multivariate discriminant analysis of the <i>UBE2D2</i> normalised gene GeXP hCellMarkerPlex data from human colon normal (white triangle) (n = 24), adenomatous polyp (grey triangle) (n = 17) and carcinoma (black triangle) (n = 19) tissues.

<p>Information on the gene symbols on the biplot is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113071#pone.0113071.s002" target="_blank">Table S2</a>. (A) Principal component analysis (PCA) biplot permits visualisation of inherent clustering patterns of individual tissue samples and associated gene expression levels. (B) Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was applied to fit a 2-class supervised model maximising covariance and discriminating gene expression profiles associated with the different tissues sample types; the biplot shows scores and loadings as well as the regression coefficients best explaining each class ($M4.DA(N),$M4.DA(A),$M4.DA(C). (C) Rank of importance of cell marker genes within the OPLS-DA. (D) Matrix showing the associated misclassification rates.</p

Localisation of <i>CDX2</i> transcripts and encoded protein in human colon.

<p>In situ hybridisation of CDX2 transcripts in human colon (A) normal, (B) adenomatous polyp and (C) carcinoma. Emulsion autoradiographs showing expression of <i>CDX2</i> in epithelium (ep) in bright field and corresponding adjacent dark field images in antisense (left) and sense (right) hybridised tissue sections (n = 5). Bar = 20 µm. (D) – (F) Representative paraffin-embedded tissue sections show immunohistochemical localisation of CDX2 expression in the human colon epithelium in (D) normal, (E) adenomatous polyp and (F) carcinoma. (G) Semi-quantitative scoring of staining intensity (increasing from + to +++) revealed increased immunostaining for CDX2 in adenomatous polyp and carcinoma (n = 8). Scoring system: + (detectable nuclear staining, weak), ++ (easily visible nuclear staining), n/a – no adenoma tissue in histological section.</p