title: Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity.
abstract: The accuracy of techniques such as microarrays, reverse transcription polymerase chain reaction, and whole transcriptome shotgun sequencing is critically dependent on RNA quality. We have repeatedly observed extensive RNA degradation following trypsinization, a routine procedure used to dissociate adherent tissue culture cells prior to RNA extraction. This study investigated the cause of this degradation and identifies an alternative procedure that enables extraction of intact high-quality RNA. Trypsinization and several alternative procedures were used to dissociate a range of different cell lines prior to RNA extraction. The contribution of exogenous ribonucleases or induction of endogenous ribonucleases by trypsin reagent proteases to RNA degradation was examined. Trypsinization resulted in a complete degradation of RNA regardless of cell line type, differentiation stage, or passage number. This occurred when intact RNA was incubated directly with trypsin and was not suppressed by inhibiting trypsin's protease activity. Prevention of degradation by sodium hypochlorite treatment of trypsin reagent identified the presence of ribonucleases in trypsin derived from animal pancreas. Consistent extraction of high-quality RNA requires the use of direct cell lysis with a phenol guanidine-based reagent or an animal origin-free protease-based dissociation agent if enzymatic detachment prior to RNA extraction cannot be avoided.
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Peter
authors.initials: P
authors.lastname: Vrtačnik
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Špela
authors.initials: Š
authors.lastname: Kos
authors.affiliation: Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford CM1 1SQ, UK.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Bustin
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Janja
authors.initials: J
authors.lastname: Marc
authors.affiliation: Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana 1000, Slovenia. Electronic address: barbara.ostanek@ffa.uni-lj.si.
authors.forename: Barbara
authors.initials: B
authors.lastname: Ostanek
mesh_terms: D045744:Cell Line, Tumor; D004587:Electrophoresis, Agar Gel; D056945:Hep G2 Cells; D006801:Humans; D061986:MCF-7 Cells; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000032:analysis / Q000302:isolation & purification; D014357:Trypsin / Q000378:metabolism*
chemical_list: D012335:RNA, Ribosomal; D012313:RNA; D014357:Trypsin
keywords: Cell culture; Cell dissociation; RNA degradation; RNA integrity; RNA quality; Trypsin