Trans-Spliced Exon Coupled RNA End Determination
Trans-Spliced Exon Coupled RNA End Determination (TEC-RED) is a transcriptomic technique that, like SAGE, allows for the digital detection of messenger RNA sequences. Unlike SAGE, detection and purification of transcripts from the 5’ end of the messenger RNA require the presence of a trans-spliced leader sequence.
Trans-splicing Background
Spliced leader sequences are short sequences of non coding
TEC-RED Methods
Total
Concatenation
Concatenation of the tags, as developed in 2004, is different from that seen in SAGE. The cleavage of the tags with Xho1 and mixture of the different samples, followed by ligation, form the first concatenation step. The second step uses one of the restriction endonucleases with consensus to the adapter molecule attached to the 3’ end. They are again ligated, and PCR is performed to purify samples for the next joining. The concatenation is continued with the second restriction endonuclease, followed by the third and finally the fourth. This results in the concatamer formed by the six endonuclease ligations containing 32 tags, arranged 5’ to 5’ around the Xho1 site.[6] In SAGE, concatenation takes place after ditags are formed and amplified by PCR. The linkers on the outside of the ditags are cleaved with the enzyme that provided their binding and these sticky end ditags are concatenated randomly and placed into a cloning vector.[7]
Advantages
The advantage of TEC-RED over SAGE is that no restriction endonuclease is needed for the initial linker binding. This prevents bias associated with restriction site sequences that will be missing from some genes, as is seen in SAGE. The ability to have a snapshot of specific RNA
Variations
Two alternate techniques have been described that allow for 5’ tag analysis in organisms that do not have trans-spliced leader sequences. The techniques presented by Toshiyuki et al. and Shin-ichi et al. are called CAGE and 5’ SAGE respectively. CAGE utilizes biotinylated cap-trapper technology to maintain mRNA signal long enough to create and select full length
See also
- RNA-seq
- DNA microarray
References
- Carninci P, Kvam C, Kitamura A, et al. (November 1996). "High-efficiency full-length cDNA cloning by biotinylated CAP trapper". Genomics. 37 (3): 327–36. PMID 8938445.
- Hashimoto S, Suzuki Y, Kasai Y, et al. (September 2004). "5'-end SAGE for the analysis of transcriptional start sites". Nat. Biotechnol. 22 (9): 1146–9. S2CID 5485989.
- Shiraki T, Kondo S, Katayama S, et al. (December 2003). "Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage". Proc. Natl. Acad. Sci. U.S.A. 100 (26): 15776–81. PMID 14663149.
- Zorio DA, Cheng NN, Blumenthal T, Spieth J (November 1994). "Operons as a common form of chromosomal organization in C. elegans". Nature. 372 (6503): 270–2. S2CID 4257343.
External links
- CAGE Tags http://genome.gsc.riken.jp/absolute/
- 5’ SAGE results https://archive.today/20040821030224/http://5sage.gi.k.u-tokyo.ac.jp/" https://archive.today/20040821030224/http://5sage.gi.k.u-tokyo.ac.jp/
- TEC RED Tags seen in wormbase https://web.archive.org/web/20080909025225/http://www.wormbase.org/db/searches/advanced/dumper