title: Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples.
abstract: BACKGROUND
  MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue.
  
  
  RESULTS
  Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p < 0.00001) and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30 < or = Ct < or = 35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance).
  
  
  CONCLUSIONS
  Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.
authors.affiliation: Division of Applied Molecular Oncology, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.
authors.forename: Rashmi S
authors.initials: RS
authors.lastname: Goswami
authors.forename: Levi
authors.initials: L
authors.lastname: Waldron
authors.forename: Jerry
authors.initials: J
authors.lastname: Machado
authors.forename: Nilva K
authors.initials: NK
authors.lastname: Cervigne
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Patricia P
authors.initials: PP
authors.lastname: Reis
authors.forename: Denis J
authors.initials: DJ
authors.lastname: Bailey
authors.forename: Igor
authors.initials: I
authors.lastname: Jurisica
authors.forename: Michael R
authors.initials: MR
authors.lastname: Crump
authors.forename: Suzanne
authors.initials: S
authors.lastname: Kamel-Reid
mesh_terms: D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000032:analysis*; D044085:Microfluidics; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012044:Regression Analysis; D015203:Reproducibility of Results
chemical_list: D035683:MicroRNAs; D005557:Formaldehyde