title: Factors in tissue handling and processing that impact RNA obtained from formalin-fixed, paraffin-embedded tissue.
abstract: Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12-24 hr in phosphate-buffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
authors: Tissue Array Research Program, Laboratory of Pathology, National Institutes of Health, Bethesda, Maryland, USA.
authors: Joon-Yong
authors: JY
authors: Chung
authors: Till
authors: T
authors: Braunschweig
authors: Reginald
authors: R
authors: Williams
authors: Natalie
authors: N
authors: Guerrero
authors: Karl M
authors: KM
authors: Hoffmann
authors: Mijung
authors: M
authors: Kwon
authors: Young K
authors: YK
authors: Song
authors: Steven K
authors: SK
authors: Libutti
authors: Stephen M
authors: SM
authors: Hewitt
mesh_terms: D000818:Animals; D002021:Buffers; D019075:Electrophoresis, Capillary; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D007668:Kidney / Q000737:chemistry; D016612:Paraffin Embedding; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling
chemical_list: D002021:Buffers; D005404:Fixatives; D012333:RNA, Messenger; D005557:Formaldehyde; D012313:RNA