title: From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues.
abstract: BACKGROUND
  Genome-wide gene expression profiling analysis of FFPE tissue samples is indispensable for cancer research and provides the opportunity to evaluate links between molecular and clinical information, however, working with FFPE samples is challenging due to extensive cross-linking, fragmentation and limited quantities of nucleic acid. Thus, processing of FFPE tissue samples from RNA extraction to microarray analysis still needs optimization.
  
  
  METHODS
  In this study, a modified deparaffinization protocol was conducted prior to RNA isolation. Trizol, Qiagen RNeasy FFPE and Arcturus PicoPure RNA Isolation kits were used in parallel to compare their impact on RNA isolation. We also evaluated the effect of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) on the percentage of present calls.
  
  
  RESULTS
  Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol gave a significantly higher percentage of present calls than the 3' IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms.
  
  
  CONCLUSIONS
  Our study paves the way for future high-throughput transcriptional analysis by optimizing FFPE tissue sample processing from RNA isolation to microarray analysis.
authors.affiliation: Ankara University, Biotechnology Institute, Ankara, Turkey.
authors.forename: Nevin
authors.initials: N
authors.lastname: Belder
authors.affiliation: Ankara University, School of Medicine, Department of Pathology, Ankara, Turkey.
authors.forename: Öznur
authors.initials: Ö
authors.lastname: Coskun
authors.affiliation: Ankara University, School of Medicine, Department of Biostatistics, Ankara, Turkey.
authors.forename: Beyza
authors.initials: B
authors.lastname: Doganay Erdogan
authors.affiliation: Middle East Technical University, Department of Statistics, Ankara, Turkey.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Ilk
authors.affiliation: Ankara University, School of Medicine, Department of Pathology, Ankara, Turkey.
authors.forename: Berna
authors.initials: B
authors.lastname: Savas
authors.affiliation: Ankara University, School of Medicine, Department of Pathology, Ankara, Turkey.
authors.forename: Arzu
authors.initials: A
authors.lastname: Ensari
authors.affiliation: Ankara University, Biotechnology Institute, Ankara, Turkey. Electronic address: hilalozdag@gmail.com.
authors.forename: Hilal
authors.initials: H
authors.lastname: Özdağ
mesh_terms: D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016612:Paraffin Embedding; D012313:RNA / Q000302:isolation & purification*; D046888:Tissue Array Analysis / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005557:Formaldehyde; D012313:RNA
keywords: FFPE tissues; Gene expression; Microarray; RNA isolation