Several methods for quantitative

analysis of the transcriptome and

gene expression
Presented by
R. Seeni Balaji
(11b223)
9/6/2014 1
(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 the
generation of expression profiles when RNA is
limited
9/6/2014 2
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 gene
expression at both the qualitative and the
quantitative level.
9/6/2014 3

Analysis of changes in expression profiles in small or scarce
biological samples, is not possible simply due to the fact
that these tissue samples do not contain the required
mRNA.

In such cases it is preferable to specifically isolate the cell
population of interest for expression profiling, rather than
using the complex tissue as a whole.

As disadvantage of SAGE is that it is characterized by a large
number of sequential reactions and purifications, which
can give rise to a significant loss of material.
9/6/2014 4
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.
9/6/2014 5
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
9/6/2014 6
LongSAGE
The method LongSAGE is an adaptation of
SAGE. Source : Saha et al. (2002)
It generates labels longer than 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.

9/6/2014 7
Complete genome annotation relies on
precise identification of transcription units
bounded by a transcription initiation site (TIS)
and a polyadenylation site (PAS).
To facilitate this process, a set of two
complementary methods, 5 Long serial
analysis of gene expression (LS) and 3LS.
The mapping of 5LS and 3LS tags to the
genome allows the localization of TIS and PAS.
9/6/2014 8

9/6/2014 9
Advantages

the annotation of genomes is even
more precise .
expression profiles obtained are extremely
accurate.
later, it benefits from new techniques
massively parallel sequencing (hundreds of
millions of tags can be analyzed).

9/6/2014 10
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 5 ends 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

9/6/2014 11

9/6/2014 12