Staphylococcus epidermidis, a normal inhabitant of a human skin and mucosa, has
emerged as one of the principle bacterial agents involved in nosocomial infections,
particularly, in patients with indwelling medical devices. It’s pathogenesis is related with
the ability to adhere and form biofilms on the surface of those medical devices and is also
associated with patients’ immune system that can be compromised. This pathologic
condition leads to a high morbidity and, uncommonly, mortality.
In the last decade, the quantification of gene expression has been one of the major
areas of research in progress. The use of molecular biology techniques, such as
quantitative (q) PCR, allowed the study of the process of biofilm formation, which is very
complex and involves elaborated genetic regulation. When determining the quantification
of genetic expression, there are two critical experimental steps that can impact the
outcome of the experiment: RNA extraction, traditionally considered the crucial step of the
whole experience, and reverse transcriptase reaction. We have previously shown that in S.
epidermidis biofilms, RNA extraction procedure has a strong influence on the outcome of
gene expression quantification. Here we evaluated the individual contributions of all
experimental steps in the outcome of reliable gene expression determinations. To achieve
that, we determined the expression of aap, fmtC and lrgB genes using the type strain
RP62A, by performing technical duplicates of each experimental step, and evaluating
thecoefficient of variability. Interestingly, our results showed that the bulk of the variability
of the gene expression quantification derived from the biological replicates, and not from
any of the experimental steps. Furthermore, variability from RNA extraction was not
significantly different from the variability obtained from reverse transcriptase or qPCR
experiments. This study further confirms that biofilms are difficult biological samples with
enhanced difficulties in gene expression determinations
