said, microarrays are still useful in the identification of differentially
regulated genes—depending on one’s experimental design, the goals
in data analysis, and resources available. The use of microarrays to
identify QS-controlled genes is described in a previous methods
chapter [1].
This chapter focuses on the use of RNAseq to identify quorum
sensing (QS)-regulated genes in prokaryotes. The main technical
challenge to consider with RNAseq is that each RNA sample will
contain large amounts of ribosomal RNA (rRNA), approximately
95% rRNA. This is problematic because one doesn’t want to per-
form RNAseq on rRNA! The goal is to perform RNAseq on the
unique transcripts in each sample, such as messenger RNAs
(mRNAs) or other RNA species. There are two main approaches
to enrich a sample for unique RNAs. In the first approach, rRNA is
removed prior to cDNA synthesis. This can be done with commer-
cially available kits that capture rRNA molecules, leaving behind
unique RNAs. These kits typically involve the use of affinity col-
umns or magnetic beads. The rRNA-depleted RNA sample can
then be used for cDNA synthesis with random hexamers. In the
second approach, one can avoid the rRNA capture step by using
not-so-random (NSR) hexamers for cDNA synthesis [2]. NSR
primer sets are depleted for hexamers that anneal to the rRNA
sequences, yet still contain hexamers that anneal to the rest of the
genome. Thus, NSR primer sets limit the amplification of rRNA
sequences. This chapter focuses on RNAseq libraries made with
NSR primers.
To identify QS-controlled genes one must compare a condition
in which QS is induced (“QS-ON”) to one in which QS is unin-
duced (“QS-OFF”). Many bacteria use QS to adapt to conditions
of high cell density. During in vitro growth, QS regulons are often
induced as bacteria leave the exponential phase of growth and enter
stationary phase. For this reason, RNAseq sample preparation from
post-exponential phase-grown cells may be a good starting point.
However, the conditions of QS induction for each organism should
be carefully considered. When selecting a sampling time for RNA-
seq, it may be prudent to first identify a QS-controlled gene or
factor and monitor it for induction.
Once the growth conditions of QS induction are established,
the appropriate control must be chosen. This can be done by
generating “QS-OFF” mutants or through signal interference (see
Fig.1 for overview). To generate “QS-OFF” mutants, inactivating
mutations may be made in the gene(s) that produce or respond to
the QS signal(s). Comparing the transcriptomes of the wild type
(WT) with the signal-negative mutant or the receptor-negative
mutant then identifies genes regulated by QS. Transcripts identified
as being differentially regulated in the comparison of the WT to the
signal-negative mutant and also in the comparison of the WT to the
receptor-negative mutant are strong candidates for targets of QS
regulation.178 Charlotte D. Majerczyk