title: Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms.
abstract: RNA quality is of utmost importance to perform gene expression quantification by qPCR. The classical methods used to determine RNA quality are based on electrophoresis and spectrophotometer assessment, namely A(260)/A(280) and A(260)/A(230) ratios. It was previously shown that due to the complex nature of Staphylococcus epidermidis biofilms, RNA extraction procedures could impact mRNA quality and thus accurate quantification. Herein, we contaminated and degraded RNA extracted from S. epidermidis biofilms, and assessed the effect on gene expression by qPCR. As expected, thermal degradation of RNA had a significant impact on gene expression on two out of the three tested genes. On the other hand, the contamination of the extracted RNA yielded an interesting result: while most contaminants did not changed the purity indicators or the integrity of RNA, significant changes on gene expression levels were found. This work confirms that poor RNA extraction has an important impact in qPCR quantification, emphasizing the consequences of carry-over contaminants on gene expression studies. Additionally, our results show that the parameters commonly used to assess the quality of extracted RNA from bacterial cultures seem to be insufficient to ensure reliable gene expression determination.
authors.affiliation: IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal.
authors.forename: Virginia
authors.initials: V
authors.lastname: Carvalhais
authors.forename: Maria
authors.initials: M
authors.lastname: Delgado-Rastrollo
authors.forename: Luís D R
authors.initials: LD
authors.lastname: Melo
authors.forename: Nuno
authors.initials: N
authors.lastname: Cerca
mesh_terms: D018441:Biofilms; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D016133:Polymerase Chain Reaction / Q000379:methods*; D020871:RNA Stability; D012329:RNA, Bacterial / Q000302:isolation & purification*; D013212:Staphylococcus epidermidis / Q000235:genetics*
chemical_list: D017931:DNA Primers; D012329:RNA, Bacterial
keywords: Impurities; RNA quality; S. epidermidis biofilm; Temperature degradation; qPCR