title: Optimized protocol for gene expression analysis in formalin-fixed, paraffin-embedded tissue using real-time quantitative polymerase chain reaction.
abstract: Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common tissue specimen available after microscopic examination. Molecular methods, such as polymerase chain reaction (PCR) and gene expression examination, serve as a source of diagnostic and prognostic information but require high-quality RNA. However, the increasing application of RNA extracted from FFPE tissue frequently results in very small and degraded quantities of nucleic acid. This study targets gene expression analysis from FFPE specimens using real-time quantitative PCR. The whole protocol consists of several steps, that is, RNA extraction and its quality control, reverse transcription, and fluorescence detection during real-time quantitative PCR. We compared several methods in each step, chose the most effective, and with that combination we successfully examined 95% (62 from 65) FFPE samples for our genes of interest. We reached the best results with RNA isolation by using a commercial kit, carefully interpreted UV spectrophotometric values, and meticulously chose reverse transcriptase and TaqMan fluorescence detection. Our protocol improves the utility of FFPE tissue for molecular profiling studies.
authors.affiliation: Laboratory of Molecular Biology, General Teaching Hospital, Prague, Czech Republic. tafka@seznam.cz
authors.forename: Hana
authors.initials: H
authors.lastname: Votavova
authors.forename: Kristina
authors.initials: K
authors.lastname: Forsterova
authors.forename: Jan
authors.initials: J
authors.lastname: Stritesky
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Velenska
authors.forename: Marek
authors.initials: M
authors.lastname: Trneny
mesh_terms: D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D016612:Paraffin Embedding; D010336:Pathology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D013048:Specimen Handling / Q000379:methods*; D014021:Tissue Preservation
chemical_list: D005404:Fixatives; D005557:Formaldehyde; D012313:RNA