Transcriptomics is the field of science that deals with the study of the

transcriptome, which is the collection of all RNA transcripts produced within

specific cells of an organism.

Transcriptomics includes the study of everything related to RNA, including its

sequences, structure, functions, locations, transcription, translation, expression

levels, and degradation. High- throughput methods, such as microarray analysis

and RNA sequencing, are used to study these RNA transcripts.

Transcriptomics is important because it includes the study of all mRNA transcripts

in a cell, which reflects actively expressed genes at any given time. By studying

RNA transcripts, transcriptomics provides information about how genetic

information is expressed, how proteins are produced, and how the cell functions.

This makes transcriptomics important for understanding how genetic information

leads to cellular activities.

The field of transcriptomics has evolved along with other fields of omics including

genomics, proteomics, and metabolomics, becoming an important part of modern

biological research. New technologies in transcriptomics have made it easier to

study RNA transcripts and their functions in different biological processes.

Historical Development of Transcriptomics

Before the development of transcriptomics, individual RNA transcripts

were studied using labor- intensive methods such as northern

blotting and Sanger sequencing. Although these techniques were popular

in the 1990 s, they could only be used to study a tiny subsection of

transcriptome and were time- consuming.

The term “transcriptome” was first used in the 1990 s. Initial attempts to

study whole transcriptomes began in 1991 with the publication of a partial

human transcriptome from the human brain.

 The early 1990 s saw the development of Expressed Sequence Tag (EST)

sequencing using Sanger sequencing.

By the mid- 1990 s, techniques like serial analysis of gene

expression (SAGE) and microarrays developed, allowing the study of

thousands of transcripts simultaneously.

RNA- Seq, developed in the mid- 20 0 0 s, further advanced the field by

allowing high- throughput sequencing of entire transcripts. Next- generation

sequencing (NGS) platforms like Illumina transformed the field of

transcriptomics.

These advancements have made it possible to study transcriptomes of

different tissues, diseases, and even single cells, contributing to our

understanding of gene regulation and cellular processes.

Types of Transcriptomics

There are different types of transcriptomics. The major types are:

1. Bulk Transcriptomics

Bulk transcriptomics studies gene expression in bulk samples containing

thousands of cells and provides a collective gene expression profile of the

entire population of cells. This method helps us to understand the gene

expression patterns in a large group of cells.

It can be used to study thousands of genes simultaneously and provides

quantitative data on gene expression levels. However, it has certain

limitations such as the inability to study cell diversity and identify rare cell

types.

W hile bulk transcriptomics includes several methods, RNA sequencing

remains the primary method to study bulk transcriptomes.

2. Single- Cell Transcriptomics

Single- cell transcriptomics is used to study the gene expression of

individual cells.

In contrast to bulk transcriptomics which studies gene expression in bulk

samples, single- cell transcriptomics measures gene expression at the

individual cell level and is useful to study the cellular diversity.

Multicellular organisms contain different types of cells, each with different

transcriptional profiles. So, the ability to study gene expression levels of

individual cells is important to understand the different cell types and

biological processes.

Single- cell transcriptomics is used to study cellular diversity and rare cell

populations that may be overlooked in bulk transcriptomics. It also allows

the discovery of novel cell types.

3. Spatial Transcriptomics

Spatial transcriptomics measures gene expression in a sample while

preserving spatial information. It is important to study the level of gene

expression along with their spatial distribution to understand the function

and structural organization of genes.

Bulk transcriptomics and single- cell transcriptomics do not provide spatial

information as they destroy the structural organization of cells.

Spatial transcriptomics technologies preserve spatial information and

provide valuable information about the organization, interactions, and

functions of cells.

Spatial transcriptomics can be divided into imaging- based and

sequencing- based technologies. Both methods are used to study

 transcriptomics while preserving spatial information.

4. Meta Transcriptomics

Meta transcriptomics studies RNA transcripts and the gene expression of

an entire community of organisms including microorganisms, plants, animals,

and other eukaryotes.

Meta transcriptomics initially focused on the study of microbial

communities only. However, it is not limited to microbes and includes other

organisms as well.

This method is useful in studying the functions and interactions of

organisms within their natural environment.

Meta transcriptomics is commonly used in environmental studies and the

 study of complex ecosystems such as microbiomes in gut, soil, and water

samples.

Methods U sed in Transcriptomics

There are different methods for transcriptome analysis. The most widely used ones

are:

1. Expressed Sequence Tag (EST) Sequencing

ESTs are short nucleotide sequences produced from a single RNA

transcript.

The process of EST sequencing begins by converting RNA into

complementary DNA (cDNA) using reverse transcription, followed by

sequencing the cDNA.

The Sanger sequencing method was the primary sequencing technique

used for ESTs until the development of high- throughput methods.

In recent years, high- throughput and large data output have made RNA-

Seq the dominant method and replaced conventional EST sequencing.

Although modern high- throughput methods have largely replaced them,

EST libraries played an important role in early microarray designs.

2. Serial Analysis of Gene Expression (SAGE)

SAGE was one of the first methods used for whole transcriptome analysis

using sequencing.

SAGE is a technique that identifies and quantifies transcripts present in a

biological sample by generating short sequence tags from mRNA.

The main principle of SAGE is to convert mRNA into short, unique

sequence tags that correspond to specific transcripts. These tags are then

concatenated and sequenced, which identifies the gene expression levels.

 SAGE involves isolating mRNA from the biological sample which is

converted into cDNA using reverse transcription. Restriction enzymes

cleave the cDNA into small fragments, which are then ligated to

oligonucleotide adapters. These fragments are further cleaved to produce

short tags, which are then ligated together to form ditags. The ditags are

concatenated into long chains, cloned into a vector for amplification, and

sequenced. The resulting sequences are analyzed to identify and quantify

the original mRNA transcripts.

SAGE provides quantitative transcriptome analysis and is useful in

discovering novel genes. However, there are several limitations of this

technique including the difficulties in identifying unknown genes due to the

short length of tags, specificity issues due to overlapping sequences, and

potential incompatibilities with some restriction enzymes.

3. Microarrays

A microarray consists of short nucleotide oligomers, called DNA probes,

which are arranged on a solid surface like a glass slide that quantifies gene

expression using hybridization.

At first, RNA is isolated from the target samples which is reverse

transcribed into cDNA and labeled with fluorescent dyes. The labeled

cDNA is hybridized to the microarray. After washing, the array is scanned

with a laser. Probes that hybridize to transcribed RNA emit light, and the

intensity of this light is used to measure gene expression levels.

Despite its ability to study gene expression and the transcriptome, the

results from microarrays can vary significantly due to differences in

laboratory procedures, laser scanners, fluorescent readouts, and analytical

methods. Therefore, microarray- based mRNA expression analysis should be

used as a preliminary step and should be validated using other methods

 including RT- PCR.

Microarrays are significantly cheaper than RNA sequencing. The data

generated by microarrays are smaller and easier to manage. However, this

method may not effectively detect genes with very low expression levels

and new transcripts.

Steps Involved in cDNA- based Microarray

RNA Sequencing (RNA- Seq)

RNA- Seq combines high- throughput sequencing with computational

methods to quantify RNA transcripts. This novel technology is based on

next- generation sequencing (NGS) platforms which are highly automated.

RNA- Seq allows simultaneous sequencing of thousands of different RNA

molecules in a single run, unlike classical sequencing methods such as

Sanger’s method.

The RNA- Seq process begins with the extraction of mRNA and converting
 it into stable cDNA. The cDNA fragments are sequenced using high-

throughput methods. The raw sequencing data are then processed to

generate a counts table. This data is used to annotate gene expression

levels. Various online tools are available for data processing.

RNA- Seq can quantify a wide range of transcripts, including novel and low-

abundance transcripts. However, RNA- Seq is much more expensive than

microarrays, making it less accessible for large- scale studies. RNA- Seq also
generates large amounts of data that require advanced bioinformatics skills

and computational resources.

Applications of Transcriptomics

Transcriptomics has applications in disease diagnosis and profiling. RNA-

Seq methods help in identifying regulatory elements like transcriptional start

sites which are important elements in human disease.

Transcriptomics is useful in understanding the molecular mechanisms of

different diseases and also provides insight into different potential

therapeutic agents.
 Transcriptomics is used in pharmacogenomics to study the response of

individuals to specific drugs and is useful in personalized medicine.

Transcriptomics is also useful in understanding host- pathogen immune

interactions and studying immune responses.

Transcriptomics is also used in developmental biology to study gene

expression during different developmental stages.

Transcriptomics is also used to study noncoding RNAs, which play

important roles in different cellular processes.