several methods for quantitative analysis of the transcriptome

8/11/2019 Several Methods for Quantitative Analysis of the Transcriptome

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Several methods for quantitative

analysis of the transcriptome and

gene expression

Presented byR. Seeni Balaji

(11b223)

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(SAGE) is a powerful expression profiling method,

allowing the analysis of the expression of

thousands of transcripts simultaneously.

A disadvantage of the method, however, is the

relatively high amount of input RNA required.

Consequently, SAGE cannot be used for thegeneration of expression profiles when RNA is

limited

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In SAGE, short sequence tags (10 bp) are

isolated from mRNA at a defined position, ligated

to long multimers, cloned and sequenced.

In addition, the short tags are long enough to

uniquely identify the corresponding transcript in

database searches.

Thus, SAGE results in an accurate picture of geneexpression at both the qualitative and the

quantitative level.

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Analysis of changes in expression profiles in small or scarcebiological samples, is not possible simply due to the factthat these tissue samples do not contain the required

mRNA.

In such cases it is preferable to specifically isolate the cellpopulation of interest for expression profiling, rather thanusing the complex tissue as a whole.

As disadvantage of SAGE is that it is characterized by a largenumber of sequential reactions and purifications, whichcan give rise to a significant loss of material.

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a modification of SAGE, named microSAGE,

which requires 500- to 5000-fold less starting

material.

Compared with SAGE, microSAGE is simplified

due to incorporation of a single-tube

procedure for all steps from RNA isolation to

tag release.

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Micro SAGE

tissue selection

RNA isolation and cDNA synthesis

Anchoring and tagging of cDNA Ligation to ditags and PCR amplification

Ditag isolation and concatenation

Sequencing and analysis of clones

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LongSAGE

The method LongSAGEis an adaptation of

SAGE.Source : Saha et al. (2002)

It generates labels longerthan 14 to 21 base

pairs and a restriction site for the

endonuclease MmeI.

Increased specificity of SAGE tags for

transcript identification and SAGE tag

mapping.

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Complete genome annotation relies onprecise identification of transcription unitsbounded by a transcription initiation site (TIS)

and a polyadenylation site (PAS). To facilitate this process, a set of two

complementary methods, 5 Long serialanalysis of gene expression (LS) and 3LS.

The mapping of 5LS and 3LS tags to thegenome allows the localization of TIS and PAS.

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Advantages

the annotationof genomes is even

more precise.

expression profiles obtained are extremelyaccurate.

later, it benefits from new techniques

massively parallel sequencing (hundreds ofmillions of tags can be analyzed).

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The CAGE method

Aims to identify TIS and promoters.

Collects 21 bp from 5 ends of cap purified cDNA.

Used in mouse and human transcriptome studies.

The method essentially uses full-length cDNAs , to the 5ends of

which linkers are attached. This is followed by the cleavage of the first 20 base pairs by class II

restriction enzymes, PCR, concatamerization, and cloning of the

CAGE tags

CAGE uses cap-trapping as the first step to capture the 5 ends of the

cDNAs, which are then transformed into short sequence (tags) of 20 to 27

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several methods for quantitative analysis of the transcriptome

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