Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol

Abstract

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used.

Assessing and Reducing Sources of Gene Expression Variability in Staphylococcus Epidermidis Biofilms

“…RNA extraction was performed as optimized previously (10). In brief, this protocol uses both chemical (phenol; AppliChem, Darmstadt, Germany) and mechanical (glass beads; Sigma-Aldrich, St. Louis, MO) lysis together with column systems for RNA isolation (E.Z.N.A Total RNA kit I, Omega Bio-Tek, Norcross, GA).…”

“…Therefore, it is important to understand the origin of such variability in order to be able to mitigate it. Variability and bias in the quantification of gene expression has been attributed to biological factors, including ( i ) RNA degradation (5), ( ii ) the presence of inhibitors (6), and ( iii ) the nature of the sample used (7), as well as non-biological factors such as ( i ) the use of different RNA extraction procedures (8), ( ii ) complementary DNA (cDNA) synthesis, and ( iii ) quantitative PCR (qPCR) kits (9, 10). Here, we measured variability associated with all three key steps in gene expression quantification: RNA extraction, cDNA synthesis, and qPCR.…”

Two Bifunctional Enzymes with Ferric Reduction Ability Play Complementary Roles during Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1

“…RNA was extracted from cells using an ultra-pure RNA rapid extraction kit (Biomed Corp., London, United Kingdom) according to the manufacturer's instructions, with some modifications; for better extraction, a bacterial sludge was created by adding liquid nitrogen and grinding the mixture into powder prior to the addition of the lysis buffer (26)(27)(28)(29). The mRNA transcripts were quantified by quantitative real-time PCR (qPCR).…”

“…qPCR was performed using a SYBR green I master kit with a LightCycler 480 qPCR instrument (Roche, South San Francisco, CA). For transcript detection, SYBR green I was added to a standard PCR mix, using 2 reaction volumes, as described previously (26). qPCR runs were performed on a CFX 96 instrument (Bio-Rad, Hercules, CA) using the cycle parameters listed in Tables S3 and S4 in the supplemental material.…”

Comparative proteomic and transcriptomic profile of Staphylococcus epidermidis biofilms grown in glucose-enriched medium

“…To ensure high quality and reliable proteomic results, an appropriate sample preparation is fundamental [19,20]. Due to the complex structure of biofilms [21], it is necessary to develop an effective lysis method in order to obtain maximum coverage of the biofilm proteome and minimal protein losses, similar to the approach optimized for total RNA extraction from S. epidermidis biofilms [22]. Different protein extraction methods, including enzymatic, chemical, mechanical and other methods available via commercial extraction kits have been tested to obtain the highest number of proteins in Staphylococcus spp.…”