Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique that generates a snapshot of mRNA populations using small tags corresponding to RNA fragments. The process involves isolating mRNA, synthesizing cDNA, and creating concatemers of tagged fragments for sequencing, allowing for detailed analysis of gene expression. Variants of the original technique, such as LongSAGE, SuperSAGE, and MACE, have been developed to improve throughput, precision, and the ability to analyze non-model organisms.

1. SERIAL ANALYSIS OF GENESERIAL ANALYSIS OF GENE
EXPRESSIONEXPRESSION ( (SAGESAGE))
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2.  Serial analysis of gene expression
(SAGE) is a transcriptomic
technique used by molecular biologists to
produce a snapshot of the messenger
RNA population in a sample of interest in
the form of small tags that correspond to
fragments of those transcripts.

3. SAGESAGE EXPERIMENTSEXPERIMENTS PROCEED AS FOLLOWS:PROCEED AS FOLLOWS:
 The mRNA of an input sample is isolated and
a reverse transcriptase and biotinylated primers
are used to synthesize cDNA from mRNA.
 The cDNA is bound to Streptavidin beads via
interaction with the biotin attached to the
primers, and is then cleaved using a restriction
endonuclease called an anchoring enzyme (AE).
The location of the cleavage site and thus the
length of the remaining cDNA bound to the
bead will vary for each individual cDNA
(mRNA).

4. PROCESS IN SAGE:PROCESS IN SAGE:

5. SAGESAGE EXPERIMENTSEXPERIMENTS PROCEED AS FOLLOWS:PROCEED AS FOLLOWS:
 The cleaved cDNA downstream from the
cleavage site is then discarded, and the
remaining immobile cDNA fragments
upstream from cleavage sites are divided in
half and exposed to one of two adapter
oligonucleotides (A or B) containing several
components in the following order upstream
from the attachment site:

6. A) Sticky ends with the anchoring enzyme
cut site to allow for attachment to cleaved
cDNA.
B) A recognition site for a restriction
endonuclease known as the tagging enzyme
(TE), which cuts about 15 nucleotides
downstream of its recognition site.
C) A short primer sequence unique to
either adapter A or B, which will later be
used for further amplification via PCR.

7.  After adapter ligation, cDNA are cleaved
using tagging enzyme (TE) to remove them
from the beads, leaving only a short "tag" of
about 11 nucleotides of original cDNA.
 The cleaved cDNA tags are then repaired
with DNA polymerase to produce blunt
end cDNA fragments.

8.  These cDNA tag fragments are ligated,
sandwiching the two tag sequences together,
and flanking adapters A and B at either end.
These new constructs, called ditags, are then
PCR amplified using anchor A and B specific
primers.
 The ditags are then cleaved using the original
anchoring enzyme, and allowed to link
together with other ditags, which will be
ligated to create a cDNA concatemer with each
ditag being separated by the Anchoring
Enzyme recognition site.

9.  These concatemers are then transformed into
bacteria for amplification through bacterial
replication.
 The cDNA concatemers can then be isolated
and sequenced using modern high-
throughput DNA sequencers and these
sequences can be analysed with computer
programs which quantify the recurrence of
individual tags.

10.  In 1995, the idea of reducing the tag length from
100 to 800 bp down to tag length of 10 to 22 bp
helped reduce the cost of mRNA surveys. In this
year, the original SAGE protocol was published
by Victor Velculescu at the Oncology Center of
Johns Hopkins University. Although SAGE was
originally conceived for use in cancer studies, it has
been successfully used to describe
the transcriptome of other diseases and in a wide
variety of organisms.

11. VICTOR VELCULESCUVICTOR VELCULESCU

12. LONG SAGELONG SAGE
 LongSAGE was a more robust version of the
original SAGE developed in 2002 which had a
higher throughput, using 20 μg of mRNA to
generate a cDNA library of thousands of
tags. Robust LongSage (RL-SAGE) Further
improved on the LongSAGE protocol with the
ability to generate a library with an insert size of 50
ng mRNA, much smaller than previous LongSAGE
insert size of 2 μg mRNA and using a lower number
of ditag polymerase chain reactions (PCR) to
obtain a complete cDNA library.

14. SUPER SAGESUPER SAGE
 SuperSAGE is a derivative of SAGE that uses
the type III-endonuclease EcoP15I
of phage P1, to cut 26 bp long sequence
tags from each transcript's cDNA, expanding
the tag-size by at least 6 bp as compared to
the predecessor techniques SAGE and
LongSAGE. The longer tag-size allows for a
more precise allocation of the tag to the
corresponding transcript, because each
additional base increases the precision of the
annotation considerably.

15.  Like in the original SAGE protocol, so-called
ditags are formed, using blunt-ended tags. By
direct sequencing with high-throughput
sequencing techniques, hundred thousands or
millions of tags can be analyzed simultaneously,
producing very precise and quantitative gene
expression profiles. Therefore, tag-based gene
expression profiling also called "digital gene
expression profiling" (DGE) can today
provide most accurate transcription profiles that
overcome the limitations of microarrays.

16. MASSIVE ANALYSIS OF CDNA ENDSMASSIVE ANALYSIS OF CDNA ENDS
 In the mid 2010s several techniques combined with
Next Generation Sequencing were developed that
employ the "tag" principle for "digital gene
expression profiling" but without the use of the
tagging enzyme. The "MACE" approach, (Massive
Analysis of cDNA Ends) generates tags
somewhere in the last 1500 bps of a transcript. The
technique does not depend on restriction enzymes
anymore and thereby circumvents bias that is
related to the absence or location of the restriction
site within the cDNA.

17.  cDNA is randomly fragmented and the
3'ends are sequenced from the 5' end of the
cDNA molecule that carries the poly-A tail.
The sequencing length of the tag can be
freely chosen. Because of this, the tags can be
assembled into contigs and the annotation of
the tags can be drastically improved.
Therefore, MACE is also use for the analyses
of non-model organisms. In addition, the
longer contigs can be screened for
polymorphisms.

18.  MACE does only require 3’ ends of
transcripts, even partly degraded RNA can be
analyzed with less degradation dependent
bias. The MACE approach uses unique
molecular identifiers to allow for
identification of PCR bias.

19. MACEMACE

20. RESULT ANALYSIS:RESULT ANALYSIS:
 The output of SAGE is a list of short sequence tags
and the number of times it is observed.
Using sequence databases a researcher can usually
determine, with some confidence, from which
original mRNA (and therefore which gene) the tag
was extracted.
 Statistical methods can be applied to tag and count
lists from different samples in order to determine
which genes are more highly expressed. For
example, a normal tissue sample can be compared
against a corresponding tumor to determine
which genes tend to be more (or less) active.