Toxicogenomics – A Drug Development Perspective - Genomic Biomarkers for Pharmaceutical Development - page 129

2. the technique can also yield information about exon junctions, allowing the study of
complex transcription units [80] ;
3. RNA-Seq has inherently low background and high sensitivity, and the upper detection
limits are not constrained, together allowing the study of the transcription across a much
wider range than for microarrays [56,81] .
A discussion of the considerable differences between available RNA-Seq technologies
is beyond the scope of this chapter. However these technologies share many common fea-
tures. First, the RNA sample is either mRNA enriched or ribosomal RNA depleted. The
choice depends on the intent of the experiment. A gene expression profiling experiment
would enrich the mRNA and ignore the other RNA species, while an experiment focused
on transcriptome characterization would deplete the ribosomal RNA leaving the mRNA,
ncRNA, miRNA, and siRNA. Next, the RNA is fragmented and size selected. The size
of RNA fragments required depends on the specific technology. Third, the fragments are
reverse-transcribed into cDNA and are clonally amplified and tagged so that they can be
attached to beads. The bead-bound fragments are then placed in a fluidics chamber, placed
in the sequencer, and sequenced. The chemistry of sequencing varies between the plat-
forms. However, each chemical change in the fluidics chamber (pH in the case of Ion Torrent,
fluorescence for the other technologies) corresponds to a specific base and the sequence is
recorded. The technologies described above all rely on the amplification of fragments via
polymerase chain reaction (PCR), which will introduce bias and change the relative propor-
tions of the RNA species present. Other technologies, referred to as 'single-molecule sequenc-
ing' or 'third-generation sequencing', avoid this amplification step and its attendant bias.
However, these technologies have not yet been widely adopted by the scientific community.
Taking all of these advantages into account, RNA-Seq represents a paradigm shift in
transcriptomics studies, with concomitant benefits for toxicogenomics. This technology has
already been extensively applied to biological research, resulting in significant and remark-
able insights into the molecular biology of cells [82-84] . The pharmaceutical industry has
already embraced sequence-based technologies, and it is likely that these technologies will
have their impact throughout the drug discovery process [85-87] .
6.3.2 Proteomics and Metabolomics
Proteomics is a methodology that attempts to compare and quantify changes of protein
profiling in a system-wide proteome analysis to evaluate the overall cellular response to drug
treatments [88,89] . Several proteomic technologies are widely used and these techniques are
complementary since they focus on subsets of proteins that are only partially overlapping.
The main distinction of these technologies resides in their being either gel-based or gel-free
techniques using liquid chromatography tandem mass spectrometry (LC-MS / MS) [90,91] .
The possibilities of proteomics and its promising results for improving current predictive and
mechanistic toxicological studies have been reviewed elsewhere [36] and [92-94] .
The majority of small molecule drugs and biologics act on protein targets. These pro-
teins do not act in isolation, but are embedded in cellular pathways and networks and
are tightly interconnected both physically and functionally with many other proteins and