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Chapter

Introductory Chapter:
Transcriptome Analysis
Miroslav Blumenberg

The central dogma of molecular biology describes the flow of genetic informa-
tion from genes to functions of the cells and organisms. This comprises a two-step
process: first, DNA, the permanent, heritable, genetic information repository, is
transcribed by the RNA polymerase enzymes into RNA, a short-lasting information
carrier; second, a subset of RNA, the messenger RNAs, mRNAs, are translated into
protein. The transcriptome, then, is the complete set of all RNA molecules in a cell,
a population of cells or in an organism.
Importantly, not all RNAs are translated into proteins, some serve a struc-
tural function, for example, rRNAs in the assembly of ribosomes, others are
transporters, e.g., tRNAs, yet others serve regulatory functions, for example, the
siRNAs, short interfering RNA, or lncRNAs, long non-coding RNAs; these are
not translated into proteins [1]. However, these non-coding RNAs can and often
do play roles in human diseases such as cancer, cardiovascular, and neurologi-
cal disorders. While transcriptomics is most commonly applied to the mRNAs,
the coding transcripts, transcriptomics also provides important data regarding
content of the cell noncoding RNAs, including rRNA, tRNA, lncRNA, siRNA, and
others. Specific approaches address the analysis of splice variant of the same gene
in different tissues.

1. Transcriptome analysis

Transcriptome Analysis is the study of the transcriptome, of the complete set of

RNA transcripts that are produced by the genome, under specific circumstances or
in a specific cell, using high-throughput methods. Transcription profiling, which
follows changes in behavior of a cell in toto, not of a single gene or just a few genes,
is used throughout diverse areas of biomedical research, including disease diagno-
sis, biomarker discovery, risk assessment of new drugs or environmental chemicals
etc. Transcription profiling can be applied to loss- and gain-of-function mutants
to identify the changes associated with the mutant phenotype. The transcriptomic
techniques have been particularly useful in identifying the functions of genes.
Transcriptomics also allows identification of pathways that respond to or ameliorate
environmental stresses. RNA-Seq can also identify disease-associated gene fusions,
single nucleotide polymorphisms and even allele-specific expression.

2. Uses of transcriptome analysis

Transcriptome Analysis is most commonly used to compare specific pairs of

samples. The differences may be due to different external environmental condi-
tions, e.g., hormonal effects or toxins. More commonly, healthy and disease states

1
Transcriptome Analysis

are compared. For example, in cancer, transcriptomics analyses address classifica-

tion, the mechanisms of pathogenesis and even outcome prediction. Transcriptome
studies can classify cancer beyond anatomical location and histopathology.
Outcome predictions can establish gene-based benchmarks to predict tumor prog-
nosis and therapy response. These approaches are already in use for personalized
medicine, individualized cancer patient therapies.
Organisms and tissues at various stages of development can be molecularly char-
acterized. The transcriptomes of stem cells help to understand the processes of cel-
lular differentiation or embryonic development. Because of its very broad approach
transcriptome analysis is a great source for identifying targets for treatment.

2.1 Methodologies

The early approach to study whole transcriptomes used microarrays, a set of

defined sequences arranged on a solid substrate [2]. Microarrays almost exclusively
represented mRNAs, that is, genes that are translated into proteins.
Nowadays the microarray approach is supplanted by high-throughput RNA
sequencing, RNA-Seq, which detects all transcripts in a sample, including the
regulatory siRNA and lncRNA transcripts [3]. In this methodology, the bulk RNA
is extracted from the sample and copied into stable double-stranded copy DNA,
ds-cDNA, which is then sequenced using various sequencing methods [4]. The
sequences obtained are aligned to reference genome sequences, available in data
banks, to identify which genes are transcribed. Quantitatively, the results provide
the expression levels for the transcribed genes. Compared to microarrays, RNA-Seq
can measure both the low-abundance and high-abundance RNAs over a five orders
of magnitude range and, importantly, RNA-Seq requires much less starting mate-
rial (nanograms vs. micrograms and even as little as 50 pg) [5]. This made possible
analyses of transcriptomes in a single cell, a great advance over bulk tissue RNA
analyses [6]. RNA-seq can be used to identify alternative splicing, novel transcripts,
and fusion genes (Table 1).
In principle, the assembly of RNA-Seq reads is not dependent on reference
genomes and can be used for gene expression studies of poorly characterized
species with limited genomic resources. It can also be used to identify novel protein
coding regions in sequenced genomes. RNA-seq can be performed using many next-
generation sequencing platforms, however, each platform has its own requirements
of sample preparation and the instrument design.

Table 1.
Comparison of RNA-seq methodologies.

2
Introductory Chapter: Transcriptome Analysis
DOI: http://dx.doi.org/10.5772/intechopen.85980

Figure 1.
Graphic representations of transcriptome analysis data. (A) Heat map with clustering tree. (B) Venn diagrams
of regulated genes.

2.2 Data analysis, repositories and presentation

Improved sequencing technologies necessitated improved data analysis methods

to deal with the increased volume of data produced by each transcriptome experi-
ment. Importantly, the results are deposited into transcriptome databases, essential
tools for transcriptome analysis. For example Gene Expression Omnibus, www.
ncbi.nml.nih.gov, contains millions of transcription profiling experiments. Such
data have potential applications beyond the original aims of an experiment. Typical
outputs include quantitative tables of the transcript levels. This requires specific
analysis algorithms, often specific to the methodology used. There are software
packages to bridge data from disparate methodologies, to identify groups of similar
expressed genes, or differentially expressed functionally significant regulatory or
metabolic pathways.

3
Transcriptome Analysis

The results of transcriptomic analyses are graphically often presented as heat

maps, a system of color-coding that represents different levels of expression of given
genes in different samples (Figure 1A). Such presentations also frequently display
a clustering of samples, this helps to identify samples with similar gene expression.
Another common graphical presentation uses Venn diagrams, which count the
transcripts which are equivalently regulated in multiple samples (Figure 1B).
Transcriptome analyses have become indispensable in basic research, transla-
tional, and clinical studies. In general, transcriptome analysis is a very powerful
hypothesis-generating tool, more than a theory proving one.

3. Specific example: transcriptome analysis applied to human skin

Easily accessible, skin was among the first targets analyzed using ‘omics’ and
dermatology embraced the approaches very early [7]. A classic example of coordi-
nated transcriptional regulation was observed in cultured fibroblasts after serum
stimulation [2]. Serum addition causes not only rapid recommencement of the cell
cycle but, characteristically a wound-healing response, a physiological role of fibro-
blasts in wound healing [8]. Transcriptional responses of epidermal keratinocytes
to UV light, hormones, vitamins, infections, inflammatory and immunomodulating
cytokines, toxins and allergens have been characterized, as were the changes associ-
ated with epidermal differentiation [9, 10].
The expression signatures that define the various cell types in human skin, were
used to define 20 specific gene signatures, including those for keratinocytes, mela-
nocytes, endothelia, adipocytes, immune cells, hair follicles, sebaceous, sweat,
and apocrine glands. This resource provided a resource named SkinSig, which was
then used to analyze 18 skin conditions, providing in-context interpretation of,
for example, influx in immune cells in inflammation or differentiation changes in
disorders of cornification [11].
In the future we can anticipate a greatly expanded usage of transcriptome
analysis. Translated to the bedside, it can provide better understanding and more
specific diagnoses of diseases. This, of course, requires additional advances in the
technology, both in the lab-bench components reducing the costs and guarantee-
ing reproducibility and accuracy, as well as in the computer-based components,
algorithms that enable physicians to establish diagnosis quickly and reliably. In a
generation, this approach will become routine.

Author details

Miroslav Blumenberg
NYU School of Medicine, USA

*Address all correspondence to: [email protected]

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

4
Introductory Chapter: Transcriptome Analysis
DOI: http://dx.doi.org/10.5772/intechopen.85980

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