Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms

https://doi.org/10.1016/j.mimet.2013.08.010Get rights and content

Highlights

  • RNA was contaminated or degraded and its effect on gene expression was assessed.

  • High temperature affected RNA integrity and gene expression.

  • Impurities affect qPCR gene expression without changes in RNA quality.

  • Common quality parameters do not relate to reliable qPCR gene expression.

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 A260/A280 and A260/A230 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.

Introduction

Staphylococcus epidermidis is one of the most prevalent species among hospitalized patients due to its ability to form strongly adherent biofilms in indwelling medical devices (Otto, 2012, Vuong and Otto, 2002, Ziebuhr et al., 2006). Transcriptional measurements are being extensively used to study S. epidermidis virulence, including biofilm formation, antibiotic resistance and also intrinsic variability between isolates (Batzilla et al., 2006, Dai et al., 2012, Fluckiger et al., 2005, Handke et al., 2004). It is well known that RNA must have high quality for downstream applications like reverse-transcriptase-quantitative PCR (qPCR), the most used technique to compare relative levels of messenger RNA (mRNA) between biological samples (Nolan et al., 2006). After extraction from bacterial cells, RNA quality including purity, integrity, and yield must be assessed (Fleige and Pfaffl, 2006). According to MIQE guidelines, RNA template should be pure, show high integrity and have sufficient quantity (Bustin et al., 2009). Even concentration is important because Sieber et al. (2010) demonstrated that when a low concentration of RNA is used, a high level of variability in gene expression is obtained.

In order to evaluate purity and concentration, ultraviolet spectroscopy data is frequently obtained. With this information, absorbance ratios 260/280 (A260/A280) and 260/230 (A260/A230) can be calculated (Manchester, 1996). To evaluate RNA integrity, the ribosomal RNA (rRNA) pattern should be analyzed by electrophoresis. Intact RNA in prokaryotic cells should present sharp bands without smearing, and the 23S rRNA should present twice the intensity of the 16S rRNA band (Pinto et al., 2009, Sambrook and Russel, 2001). However, in the last few years, a methodology based on a microfluidic capillary electrophoresis system has been increasingly used to quantify and determine RNA integrity, such as 2100 Bioanalyzer (Agilent Technologies) and Experion (Bio-Rad) (Riedmaier et al., 2010). Nevertheless, the combination of agarose gel electrophoresis and NanoDrop™ spectrophotometer is still used most of the time due to simplicity and cost-effective balance (Jahn et al., 2008). Furthermore, RNA Integrity Number (RIN) and RNA quality number (RQI) thresholds for high quality prokaryotic total RNA weren't defined yet, however, a RIN above 7 has been accepted as appropriate for qPCR (Jahn et al., 2008).

As demonstrated by some studies, different RNA extraction methods can yield RNA with distinct quality, and this has been associated with the complex nature of biological samples (Atshan et al., 2012, Franca et al., 2011, Franca et al., 2012a, Pinto et al., 2009). Moreover, different components from biological samples (Radstrom et al., 2004, Tichopad et al., 2004) or from the RNA isolation procedure used, such as ethanol, isopropanol, phenol and many salts can inhibit the PCR process (Bar et al., 2012, Radstrom et al., 2004, Wilson, 1997). In addition, temperature is also a known factor which interferes with the preservation of RNA integrity (Opitz et al., 2010, Sung et al., 2003).

Franca et al. (2012a) have recently shown that mRNA quantification in bacterial biofilms was highly variable depending on the RNA extraction kit used, and RNA quality indicators did not always correlate with a reliable gene expression quantification. This is of significant importance since the most used quality indicators that should guarantee RNA quality seem not to be enough to determine the reliability of bacterial gene expression (Lloyd et al., 2010). In this study, we aimed to assess how induced RNA degradation, by temperature and controlled contamination of RNA, affected quality parameters and their consequences in gene expression of S. epidermidis biofilms.

Section snippets

Biofilm growth conditions

Biofilm forming S. epidermidis 9142 was used as a model strain. One colony was inoculated in Tryptic Soy Broth (TSB) (Oxoid) and incubated at 37 °C in a shaker at 120 rpm overnight. Then, a 1:100 dilution was performed in TSB enriched with 0.4% (w/v) glucose (Fisher Scientific) in a 24-well plate and was incubated in the same conditions. After 24 h, biofilms were washed with phosphate buffer solution (PBS) diluted 1:3 (180 mM NaCl, 3 mM KCl, 9 mM Na2HPO4·2H2O, 1.5 mM KH2PO4) and suspended in 1 mL of

RNA degradation

It is widely accepted that RNA with high purity and integrity is of extreme importance to guarantee a reliable analysis of gene expression (Vermeulen et al., 2011). However, it has also been shown that bacterial gene expression variability does not always correlate with high quality RNA, as determined by the standard parameters of RNA quality (Franca et al., 2012a). Based on the analysis of agarose gel electrophoresis and NanoDrop measurement, we assessed the impact of RNA quality on qPCR gene

Conclusions

As expected, RNA that showed loss of integrity had different genetic expression in some genes. However, despite no visible loss of rRNA integrity and no statistically significant alterations in NanoDrop parameters, contamination with WBII also strongly influenced gene expression.

This work suggests that while the electrophoresis band pattern analysis and NanoDrop quality parameters can reveal physical characteristics of RNA, they are not enough to determine the reliability and stability of gene

Acknowledgments

This work was funded by the Fundacão para a Ciência e a Tecnologia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309. The following authors had an individual FCT fellowship: VC (SFRH/BD/78235/2011); LDRM (SFRH/BD/66166/2009).

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