Author(s): Sousa, Cármen ; França, Ângela ; Cerca, Nuno
Date: 2014
Persistent ID: http://hdl.handle.net/1822/31922
Origin: RepositóriUM - Universidade do Minho
Subject(s): biofilms; gene expression variability; biofilm pool; RNA extraction; reverse transcriptase; qPCR; Science & Technology
Gene expression quantification can be a useful tool in studying the properties of bacterial biofilms. Unfortunately, techniques such as RNA extraction, cDNA synthesis, and quantitative PCR (qPCR) can introduce variability into mRNA transcript measurements, obscuring biologically relevant results. Here we sought to identify the steps that impair accurate gene expression quantification from Staphylococcus epidermidis biofilm samples. We devised an experimental setup that could be used to determine the contribution of each experimental step to the variability of mRNA transcript measurement. Among factors tested, biofilm growth contributed the most bias to gene expression quantification. Additional experiments demonstrated that pooling biofilms together reduced this variability, resulting in more accurate gene expression analysis results. We therefore recommend pooling in order to reduce the variability associated with gene expression quantification from biofilm samples.
We would like to thank Kimberly K. Jefferson at Virginia Commonwealth University for reviewing the manuscript. This work was co-funded by Fundacao para a Ciencia e a Tecno logia (FCT) and COMPETE grants PTDC/BIA-MIC/113450/2009 and FCOMP-01-0124-FEDER-014309, FCT Strategic Project PEst-OE/EQB/LA0023/2013, and FCT project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and by OREN, FEDER, ON2 project. NORTE-07-0124-FEDER-000027. N.C. is an Investigador FCT.
