Preparation of archival formalin-fixed paraffin-embedded mouse liver samples for use with the Agilent gene expression microarray platform

J Pharmacol Toxicol Methods. Sep-Oct 2013;68(2):260-268. doi: 10.1016/j.vascn.2013.02.008. Epub 2013 Mar 1.

Abstract

Introduction: Tissue samples are routinely formalin-fixed and paraffin-embedded (FFPE) for long term preservation. Gene expression analysis of archival FFPE tissues may advance knowledge of the molecular perturbations contributing to disease. However, formalin causes extensive degradation of RNA.

Methods: We compared RNA quality/yield from FFPE samples using six commercial FFPE RNA extraction kits. In addition we compared four DNA microarray protocols for the Agilent 8×60K platform using 16year old FFPE mouse liver samples treated with phenobarbital or vehicle.

Results: Despite low quality RNA, archival phenobarbital samples exhibited strong induction of the positive control genes Cyp2b9 and Cyp2b10 by quantitative real-time PCR (qPCR). We tested one- and two-color microarray designs and evaluated the effects of increasing the amount of hybridized cDNA. Canonical gene responders to phenobarbital were measurably induced under each experimental condition. Increasing the amount of labeled cDNA did not improve the overall signal intensity. One-color experiments yielded larger fold changes than two-color and the number of differentially expressed genes varied between protocols. Gene expression changes were validated by qPCR and literature searches. Individual protocols exhibited high rates of false positives; however, pathway analysis revealed that nine of the top ten canonical pathways were consistent across experiments. Genes that were differentially expressed in more than one experiment were more likely to be validated. Thus, we recommend that experiments on FFPE samples be done in duplicate to reduce false positives.

Discussion: In this analysis of archival FFPE samples we were able to identify pathways that are consistent with phenobarbital's mechanism of action. Therefore, we conclude that FFPE samples can be used for meaningful microarray gene expression analyses.

Keywords: 1.5X; 1C; 1X; 2C; Archival tissue; CAR; Cy; Cyp; DEG; FF; FFPE; Formalin-fixed paraffin-embedded (FFPE); Gene expression; IPA; Ingenuity Pathway Analysis; K; LOWESS; LincRNA; MAANOVA; Microarray (Agilent); PCR; PXR; Phenobarbital; RIN; RNA; RNA integrity number; RXR; SD; ULS; Universal Linkage System; WTA; cDNA; complementary deoxyribonucleic acid; constitutive androstane receptor; cyanine; cytochrome P450; dT; deoxy-thymine; differentially expressed gene; formalin-fixed paraffin-embedded; fresh-frozen; high cDNA input; locally weighted scatterplot smoothing; long intergenic non-coding RNA; microarray analysis of variance; normal cDNA input; oligo; oligonucleotide; one-color; polymerase chain reaction; pregnane X receptor; qPCR; quantitative PCR; retinoid X receptor; ribonucleic acid; standard deviation; thousand; two-colour; whole transcriptome amplification.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases / genetics
  • Cytochrome P450 Family 2
  • False Positive Reactions
  • Formaldehyde / chemistry
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation / drug effects*
  • Liver / metabolism*
  • Male
  • Mice
  • Mice, Transgenic
  • Oligonucleotide Array Sequence Analysis / methods
  • Paraffin Embedding / methods
  • Phenobarbital / pharmacology
  • Real-Time Polymerase Chain Reaction
  • Steroid Hydroxylases / genetics
  • Tissue Fixation / methods*

Substances

  • Formaldehyde
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Cyp2b10 protein, mouse
  • Cyp2b9 protein, mouse
  • Cytochrome P450 Family 2
  • Phenobarbital