Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR

Diagn Mol Pathol. 2008 Jun;17(2):101-6. doi: 10.1097/PDM.0b013e318163f26e.

Abstract

The determination of marker genes and gene clusters involved in disease pathogenesis is increasingly contingent on high-throughput methods of gene expression profiling. However, the concurrently increasing application of mRNA from formalin-fixed and paraffin-embedded (FFPE) tissue archives, as well as cell-type-specific approaches by laser-assisted microdissection, frequently results in very small and degraded quantities of RNA. Therefore, a successful amplification of cell-type-specific mRNA targets from FFPE tissues becomes more and more essential. To optimize the hitherto limited technical options, we applied 3 commercial amplification kits on FFPE single cells. We thereby determined the approach of target-specific cDNA amplification as being notably appropriate for subsequent real-time polymerase chain reaction, as a constant decrease of CT values by 14 polymerase chain reaction cycles could be demonstrated.

Publication types

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

MeSH terms

  • Bone Marrow Cells / chemistry
  • Bone Marrow Cells / pathology
  • Fixatives / chemistry
  • Formaldehyde / chemistry
  • Gene Amplification
  • Gene Expression Profiling / methods*
  • Humans
  • Lasers
  • Microdissection
  • Neoplasms / chemistry
  • Neoplasms / genetics
  • Paraffin Embedding
  • RNA, Messenger / genetics*
  • RNA, Neoplasm / analysis
  • RNA, Neoplasm / genetics*
  • Reagent Kits, Diagnostic*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Tissue Fixation

Substances

  • Fixatives
  • RNA, Messenger
  • RNA, Neoplasm
  • Reagent Kits, Diagnostic
  • Formaldehyde