Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis

PLoS One. 2013 Jul 31;8(7):e70714. doi: 10.1371/journal.pone.0070714. Print 2013.

Abstract

Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryopreservation / methods*
  • Cryopreservation / standards
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / genetics
  • Formaldehyde / chemistry
  • Humans
  • Oligonucleotide Array Sequence Analysis / methods
  • Oligonucleotide Array Sequence Analysis / standards
  • Paraffin Embedding / methods*
  • Paraffin Embedding / standards
  • Quality Control
  • RNA / analysis*
  • RNA / genetics
  • RNA / isolation & purification*
  • RNA Stability
  • Reference Standards
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Tissue Fixation / methods*
  • Tissue Fixation / standards

Substances

  • DNA, Complementary
  • Formaldehyde
  • RNA

Grant support

This work was supported by the following grants: SPIDIA (222916) (http://www.spidia.eu/); Austrian Genome Program GEN-AU (J20964) http://www.gen-au.at/index.jsp?lang=en. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.