title: The importance of reference gene analysis of formalin-fixed, paraffin-embedded samples from sarcoma patients - an often underestimated problem.
abstract: OBJECTIVE
  Reverse transcription quantitative real-time polymerase chain reaction is efficient for quantification of gene expression, but the choice of reference genes is of paramount importance as it is essential for correct interpretation of data. This is complicated by the fact that the materials often available are routinely collected formalin-fixed, paraffin-embedded (FFPE) samples in which the mRNA is known to be highly degraded. The purpose of this study was to investigate 22 potential reference genes in sarcoma FFPE samples and to study the variation in expression level within different samples taken from the same tumor and between different histologic types.
  
  
  METHODS
  Twenty-nine patients treated for sarcoma were enrolled. The samples encompassed 82 (FFPE) specimens. Extraction of total RNA from 7-μm FFPE sections was performed using a fully automated, bead-base RNA isolation procedure, and 22 potential reference genes were analyzed by reverse transcription quantitative real-time polymerase chain reaction. The stability of the genes was analyzed by RealTime Statminer. The intrasamples variation and the interclass correlation coefficients were calculated. The linear regression model was used to calculate the degradation of the mRNA over time.
  
  
  RESULTS
  The quality of RNA was sufficient for analysis in 84% of the samples. Recommended reference genes differed with histologic types. However, PPIA, SF3A1, and MRPL19 were stably expressed regardless of the histologic type included. The variation in ∆Cq value for samples from the same patients was similar to the variation between patients. It was possible to compensate for the time-dependent degradation of the mRNA when normalization was made using the selected reference genes.
  
  
  CONCLUSIONS
  PPIA, SF3A1, and MRPL19 are suitable reference genes for normalization in gene expression studies of FFPE samples from sarcoma regardless of the histology.
authors.affiliation: Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark. Electronic address: aggerholm@oncology.dk.
authors.forename: Ninna
authors.initials: N
authors.lastname: Aggerholm-Pedersen
authors.affiliation: Department of Oncology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Akmal
authors.initials: A
authors.lastname: Safwat
authors.affiliation: Department of Pathology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Steen
authors.initials: S
authors.lastname: Bærentzen
authors.affiliation: Department of Oncology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Marianne
authors.initials: M
authors.lastname: Nordsmark
authors.affiliation: Faculty of Health, Aarhus University, Aarhus, Denmark.
authors.forename: Ole Steen
authors.initials: OS
authors.lastname: Nielsen
authors.affiliation: Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Jan
authors.initials: J
authors.lastname: Alsner
authors.affiliation: Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Brita S
authors.initials: BS
authors.lastname: Sørensen