Serial analysis of gene expression (SAGE) is a powerful technique that uses mRNA from a particular sample to generate complementary DNA (cDNA) fragments which are then amplified and sequenced using high-throughput sequencing technology.
The basic mechanism behind SAGE is based on tags which can identify the original transcript, and fast sequencing of chains of tags linked together. The procedure essentially simplifies sequencing by linking the cDNA segments together to form a long chain.
Steps of SAGE
- mRNA is separated from the sample and reverse transcribed using biotinylated primers to produce cDNA
- cDNA is bound via biotin to streptavidin microbeads
- cDNA is cut with restriction enzymes freeing it from the beads
- Cleaved DNA is washed out, leaving truncated cDNA bound to the beads
- Two oligonucleotides with sticky ends are added to the remaining truncated cDNA, in separate samples
- Cleaved DNA is “tagged” enzymatically, removing it from the beads
- Sticky ends are further repaired with DNA polymerase
- Blunt ended tags from the two separate samples are ligated together, generating di-tags with two different oligonucleotide adapter ends
- Di-tags are cleaved to remove the oligonucleotides.
- Transform concatemers into bacteria for replication
- Separate concatemers from bacteria and sequence
SAGE is similar in many ways to a DNA microarray; however, in a DNA microarray, the mRNAs hybridize to cDNA probes on the array. In SAGE, the output data is based on sequencing. That means SAGE analysis is more quantitative and it does not depend on the use of known genes. Microarray experiments are generally less costly, and so are used more often in larger-scale studies.
- Genome wide analysis of gene expression
- New markers are studied in cancer by using SAGE
- Researchers compared gene expression levels in cancerous tissues with those in non-cancerous tissues to search for markers that could diagnose the pancreatic cancer at an early stage
- Researchers are able to identify a gene called prostate stem cell antigen (PCSA) in cancer problems
The SAGE technique measures a ‘tag’ which shows the transcriptome product of a gene. A tag for the purpose of SAGE is a nucleotide sequence of a defined length, directly adjacent to the 3’-most restriction site for a particular restriction enzyme. Thus, the data product of SAGE is a list of tags with their count values, giving a digital representation of cellular gene expression. Certain technical modifications have been made to the original SAGE protocol to improve its efficiency, reducing the amount of input RNA, increasing the length of SAGE tags, and allowing further use of SAGE results.
In plants, SAGE has been used to analyze host-pathogen interactions, plant responses to many environmental and nutritional stresses, metabolism of toxic compounds and transcript profiling of a particular tissue/organ.