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Open Access Research

Multiplex transcriptional analysis of paraffin-embedded liver needle biopsy from patients with liver fibrosis

Nicholas R Staten15*, Eric A Welsh1, Kurex Sidik1, Sandra A McDonald1, Dawn R Dufield1, Botoul Maqsodi2, Yunqing Ma2, Gary K McMaster2, Rodney W Mathews1, Robert H Arch3, Jaime L Masferrer1 and Bernard E Souberbielle46*

Author Affiliations

1 Pfizer Global Research & Development, 700 Chesterfield Parkway West, Chesterfield, MO, 63017, USA

2 Affymetrix, 3380 Central Expressway, Santa Clara, CA, 95051, USA

3 Pfizer Worldwide Research & Development, Asia Research 575 Maryville Centre Drive, Saint Louis, MO, 63141, USA

4 Pfizer Clinical Research, Ramsgate Road, Sandwich, Kent, CT139NJ, UK

5 Present address: Kypha, Inc., 4320 Forest Park Avenue, St Louis, MO, 63108, USA

6 Present address: GSK, Experimental Medicine Unit, Gunnels Wood Road, Stevenage, Herst, SG1 2NY, UK

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Fibrogenesis & Tissue Repair 2012, 5:21  doi:10.1186/1755-1536-5-21

Published: 27 December 2012

Abstract

Background

The possibility of extracting RNA and measuring RNA expression from paraffin sections can allow extensive investigations on stored paraffin samples obtained from diseased livers and could help with studies of the natural history of liver fibrosis and inflammation, and in particular, correlate basic mechanisms to clinical outcomes.

Results

To address this issue, a pilot study of multiplex gene expression using branched-chain DNA technology was conducted to directly measure mRNA expression in formalin-fixed paraffin-embedded needle biopsy samples of human liver. Twenty-five genes were selected for evaluation based on evidence obtained from human fibrotic liver, a rat BDL model and in vitro cultures of immortalized human hepatic stellate cells. The expression levels of these 25 genes were then correlated with liver fibrosis and inflammation activity scores. Statistical analysis revealed that three genes (COL3A1, KRT18, and TUBB) could separate fibrotic from non-fibrotic samples and that the expression of ten genes (ANXA2, TIMP1, CTGF, COL4A1, KRT18, COL1A1, COL3A1, ACTA2, TGFB1, LOXL2) were positively correlated with the level of liver inflammation activity.

Conclusion

This is the first report describing this multiplex technique for liver fibrosis and has provided the proof of concept of the suitability of RNA extracted from paraffin sections for investigating the modulation of a panel of proinflammatory and profibrogenic genes. This pilot study suggests that this technique will allow extensive investigations on paraffin samples from diseased livers and possibly from any other tissue. Using identical or other genes, this multiplex expression technique could be applied to samples obtained from extensive patient cohorts with stored paraffin samples in order to correlate gene expression with valuable clinically relevant information. This method could be used to provide a better understanding of the mechanisms of liver fibrosis and inflammation, its progression, and help development of new therapeutic approaches for this indication.

Keywords:
Liver fibrosis; Liver inflammation; Multiplex gene expression