role of transcriptomics in gene expression studies and

Role of transcriptomics in gene expression studies and its relevance

Name: Sarla YadavClass: M.Sc.MBT 3rd sem

Roll No.: 1873

Introduction

• Transcriptome: – The transcriptome is the complete set of transcripts

in a cell, both in terms of type and quantity.• Transcriptomics:– The global studies of gene expression at the RNA

level.– Comparison of transcriptomes allows the

identification of genes that are differentially expressed in distinct cell population, or in response to different treatment.

Transcriptome: An evolving definition

• (the population of) mRNAs expressed by a genome at any given time(Abbott, 1999)• The complete collection of transcribed elements of the genome. (Affymetrix, 2004)

mRNAs Non-coding RNAs

tRNAs rRNAs snmRNAs (small non-messenger RNAs)

microRNAs and siRNAs snoRNAs (small nucleolar RNAs) snRNAs (small nuclear RNAs)

Pseudogenes

Transcriptome

• Transcriptome is much more than just the transcribed portion of the genome..

• A gene may produce many different types of mRNA molecules

• Hence it is highly dynamic and complex.• Complexity of transcriptome is increased by processes

such as– Alternate splicing– RNA editing

Central dogma of life

“transcripts”

Alternative splicing adds to the size (complexity) of the transcriptome.

RNA editing adds to the size (complexity) of the transcriptome.

Methods of observing gene expression

• Focussed Experimental Approaches:

--Northern Blotting Analysis

--Real time PCR (quantitative or semi-quantitative)

• Highthroughput Approaches

– Microarray expression profiling

– Serial analysis of gene expression (SAGE)

– RNA sequencing

Northern Blotting Analysis

Real time PCR (quantitative or semi-quantitative)

Microarray expression profiling

SAGE

RNA sequencingIllumina HiSeq2000/2500Bridge amplification, clonal expansion

RNA-sequencing

• Recent advances in sequencing technologies have made possible the comprehensive and in-depth characterization of transcriptomes via a technique known as RNA-Seq

• Compared to the use of microarrays, RNA-Seq is able to quantify more RNA species, including non-coding and novel splice variants,

Comparison of Methods for Studying the Transcriptome

Role of transcriptomics

• Reveal the process of development• Determine the role of ncRNAs (miRNA)• Genetic basis of diseases• Help in study the response of drug

Transcriptomics in diseases diagnosis

• Whole transcriptome sequencing reveals that alzheimer’s patient have different gene expression splicing pattern in brain regions.

• It affects individuals over the age of 60• It is characterized by a complex progression of neurodegeneration

that results in – memory impairment – loss of other cognitive processes as well as the presence of non-

cognitive symptoms including delusions, agitation and changes in mood and personality.

Transcriptomics in diseases diagnosis

• A comparative gene expression analysis of normal human brain tissue and tissue affected by Alzheimer’s disease, using the RNA-Seq technique.

• Along with samples from whole normal and AD brains, mRNA samples from two different brain regions, namely the frontal and temporal lobes, were analyzed.

• We found significant differences in gene isoform expression levels, alternated use of promoters and transcription start sites between normal and AD brain tissue

Transcriptomics in the analysis of drug response

• Asthma, a chronic inflammatory respiratory disease that is characterized by variable airflow limitation and airway hyper responsiveness.

• Glucocorticoids (GCs) are common medications used to treat various inflammatory diseases, including asthma.

• The mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood

• Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone—a potent synthetic glucocorticoid (1 mM for 18 hours).

One stretch of DNA can encode an mRNA together with many other types of RNAs.

Analysis the role of ncRNAs via transcriptomics

• Transcriptomics also reveal the regulatory function of ncRNAs such as miRNA.

microRNAs are functional gene products 21 – 22 nucleotides long

miRNA functions

microRNAs are mis-regulated in human cancers.

This is important for cancer diagnosis.

miRNA responsible for cancer

• Specific miRNA can be upregulated in cancer and global miRNA downregulation is a common trait of human malignancies

• In human breast cancer high level of miR103/107 are associated to metastasis

• At cellular level miR103/107 is responsible for the induction of epithelial to mesenchymal transition (EMT) attained by downregulation miR-200 levels.

EMT

microRNAs are involved in cancer metastasis

miRNAs are involved in cancer metastasis

Role in treatment of diseases

• By trancriptome analysis it revealed that hepatocellular carcinoma cells exhibit reduce expression of miR-26a, a miRNA that normally expressed at high levels in diverse tissues.

microRNAs can be used to treat liver cancer (in mice).

microRNA miR-26a

Thank you