Retrospective microRNA sequencing: Complementary DNA library preparation protocol using formalin-fixed paraffin-embedded RNA specimens

Olivier Loudig, Christina Liu, Thomas E. Rohan, Iddo Z. Ben-Dov

Research output: Contribution to journalArticle

Abstract

Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered froFFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18–24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3' barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3' barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Original languageEnglish (US)
Article numbere57471
JournalJournal of Visualized Experiments
Volume2018
Issue number135
DOIs
StatePublished - May 5 2018

Keywords

  • CDNA library
  • Degraded RNA
  • Degraded RNA
  • FFPE RNA
  • Formalin-fixed paraffin-embedded
  • Genetics
  • Issue 135
  • MicroRNA
  • Next-generation sequencing
  • PAGE
  • PCR amplification
  • Quantification
  • T4 RNA ligation

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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