Scalable in Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers

PLoS One. 2012;7(3):e32927. doi: 10.1371/journal.pone.0032927. Epub 2012 Mar 8.

Abstract

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / metabolism
  • Biomarkers, Tumor / genetics*
  • Biomarkers, Tumor / metabolism
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / metabolism
  • Colorectal Neoplasms / pathology
  • Humans
  • Immunohistochemistry
  • In Situ Hybridization / methods*
  • Matrix Attachment Region Binding Proteins / genetics
  • Matrix Attachment Region Binding Proteins / metabolism
  • Neoplasms / genetics*
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Organ Specificity / genetics
  • Reproducibility of Results
  • Tissue Array Analysis / methods*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Biomarkers
  • Biomarkers, Tumor
  • Matrix Attachment Region Binding Proteins
  • SATB2 protein, human
  • Transcription Factors