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Anal Biochem. 2014 Oct 15;463:38-44. doi: 10.1016/j.ab.2014.06.017. Epub 2014 Jun 28.

Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity.

Author information

1
Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana 1000, Slovenia.
2
Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford CM1 1SQ, UK.
3
Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana 1000, Slovenia. Electronic address: barbara.ostanek@ffa.uni-lj.si.

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.

KEYWORDS:

Cell culture; Cell dissociation; RNA degradation; RNA integrity; RNA quality; Trypsin

PMID:
24983903
DOI:
10.1016/j.ab.2014.06.017
[Indexed for MEDLINE]

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