Transcriptomics technologies: Difference between revisions

Revision as of 14:16, 24 October 2022

Revision as of 14:17, 24 October 2022

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===Serial and cap analysis of gene expression (SAGE/CAGE)===

[[File:Summary of SAGE.svg|thumb|500pxupright=2|''Summary of [[Serial analysis of gene expression|SAGE]].'' Within the organisms, genes are [[Transcription (biology)|transcribed]] and [[RNA splicing|spliced]] (in [[eukaryote]]s) to produce mature [[Messenger RNA|mRNA]] transcripts (red). The mRNA is extracted from the organism, and [[reverse transcriptase]] is used to copy the mRNA into stable double-stranded–cDNA ([[Nucleic acid double helix|ds]]-[[Complementary DNA|cDNA]]; blue). In SAGE, the ds-cDNA is digested by [[restriction enzyme]]s (at ___location ‘X’ and ‘X’+11) to produce 11-nucleotide "tag" fragments. These tags are concatenated and sequenced using long-read [[Sanger sequencing]] (different shades of blue indicate tags from different genes). The sequences are [[Deconvolution|deconvoluted]] to find the frequency of each tag. The tag frequency can be used to report on [[Transcription (biology)|transcription]] of the gene that the tag came from.<ref name="Lowe_2017">{{cite journal | vauthors = Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T | title = Transcriptomics technologies | journal = PLOS Computational Biology | volume = 13 | issue = 5 | pages = e1005457 | date = May 2017 | pmid = 28545146 | pmc = 5436640 | doi = 10.1371/journal.pcbi.1005457 | bibcode = 2017PLSCB..13E5457L }}</ref>]]

[[Serial analysis of gene expression]] (SAGE) was a development of EST methodology to increase the throughput of the tags generated and allow some quantitation of transcript abundance.<ref name="#7570003" /> [[Complementary DNA|cDNA]] is generated from the [[RNA]] but is then digested into 11&nbsp;bp "tag" fragments using [[restriction enzyme]]s that cut DNA at a specific sequence, and 11&nbsp;base pairs along from that sequence. These cDNA tags are then [[Ligation (molecular biology)|joined]] head-to-tail into long strands (>500&nbsp;bp) and sequenced using low-throughput, but long read-length methods such as [[Sanger sequencing]]. The sequences are then divided back into their original 11 bp tags using computer software in a process called [[deconvolution]].<ref name="#7570003" /> If a high-quality [[reference genome]] is available, these tags may be matched to their corresponding gene in the genome. If a reference genome is unavailable, the tags can be directly used as diagnostic markers if found to be [[Gene expression profiling|differentially expressed]] in a disease state.<ref name="#7570003" />