title: Evaluation of commercial kits for extraction of DNA and RNA from Clostridium difficile.
abstract: Commercial nucleic acid extraction kits are a cost effective, efficient and convenient way to isolate DNA and RNA from bacteria. Despite the increasing importance of the gastrointestinal pathogen, Clostridium difficile, and the increased use of nucleic acids in its identification, characterization, and investigation of virulence factors, no standardized or recommended methods for nucleic acid isolation exist. Here, we sought to evaluate 4 commercial DNA extraction kits and 3 commercial RNA extraction kits assessing cost, labor intensity, purity, quantity and quality of nucleic acid preparations. The DNA extraction kits produced a range of concentrations (20.9-546 ng/ml) and A(260/280) ratios (1.92-2.11). All kits were suitable for DNA extraction with the exception of the Roche MagNA pure LC DNA isolation kit III which produced DNA of high yield but with substantial shearing, but that did not affect downstream PCR amplifications. For RNA extraction, the Qiagen RNeasy mini kit stood out producing preparations of consistently higher concentrations and higher RNA integrity numbers (RIN). The Roche MagNA pure LC RNA isolation kit produced preparations that could not be properly assigned RINs due to a failure to remove small RNAs which were interpreted as degradation. Good DNA and RNA yield are critical but methods are often overlooked. This study highlights the potential for critical variation between established commercial systems and the need for assessment of any extraction methods that are used.
authors.affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G 2W1. dmetcalf@uoguelph.ca
authors.forename: Devon
authors.initials: D
authors.lastname: Metcalf
authors.forename: J Scott
authors.initials: JS
authors.lastname: Weese
mesh_terms: D016360:Clostridioides difficile / Q000235:genetics*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D008967:Molecular Biology / Q000379:methods*; D012329:RNA, Bacterial / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial