Transcriptomics technologies: Difference between revisions

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|100,000:1 (limited by sequence coverage)<ref name="#24194394">{{cite journal | vauthors = Black MB, Parks BB, Pluta L, Chu TM, Allen BC, Wolfinger RD, Thomas RS | title = Comparison of microarrays and RNA-seq for gene expression analyses of dose-response experiments | journal = Toxicological Sciences | volume = 137 | issue = 2 | pages = 385–403 | date = February 2014 | pmid = 24194394 | doi = 10.1093/toxsci/kft249 | doi-access = free }}</ref>

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The dominant contemporary techniques, [[DNA microarray|microarrays]] and [[RNA-Seq]], were developed in the mid-1990s and 2000s.<ref name="#23290152" /><ref name="#11287436">{{cite journal | vauthors = Nelson NJ | title = Microarrays have arrived: gene expression tool matures | journal = Journal of the National Cancer Institute | volume = 93 | issue = 7 | pages = 492–4 | date = April 2001 | pmid = 11287436 | doi = 10.1093/jnci/93.7.492 }}</ref> Microarrays that measure the abundances of a defined set of transcripts via their [[Nucleic acid hybridization|hybridisation]] to an array of [[Complementarity (molecular biology)|complementary]] [[Molecular probe|probes]] were first published in 1995.<ref name="#7569999">{{cite journal | vauthors = Schena M, Shalon D, Davis RW, Brown PO | title = Quantitative monitoring of gene expression patterns with a complementary DNA microarray | journal = Science | volume = 270 | issue = 5235 | pages = 467–70 | date = October 1995 | pmid = 7569999 | doi = 10.1126/science.270.5235.467 | bibcode = 1995Sci...270..467S | s2cid = 6720459 }}</ref><ref name="#17644526">{{cite journal | vauthors = Pozhitkov AE, Tautz D, Noble PA | title = Oligonucleotide microarrays: widely applied—poorly understood | journal = Briefings in Functional Genomics & Proteomics | volume = 6 | issue = 2 | pages = 141–8 | date = June 2007 | pmid = 17644526 | doi = 10.1093/bfgp/elm014 | url = http://edoc.mpg.de/get.epl?fid=85687&did=572223&ver=0 | doi-access = free | hdl = 11858/00-001M-0000-000F-D7B3-3 | hdl-access = free }}</ref> Microarray technology allowed the assay of thousands of transcripts simultaneously and at a greatly reduced cost per gene and labour saving.<ref name=pmid12117754>{{cite journal | vauthors = Heller MJ | title = DNA microarray technology: devices, systems, and applications | journal = Annual Review of Biomedical Engineering | volume = 4 | pages = 129–53 | year = 2002 | pmid = 12117754 | doi = 10.1146/annurev.bioeng.4.020702.153438 }}</ref> Both [[DNA microarray#Spotted vs. in situ synthesised arrays|spotted oligonucleotide arrays]] and [[Affymetrix]] high-density arrays were the method of choice for transcriptional profiling until the late 2000s.<ref name="#19715439" /><ref name="#11287436" /> Over this period, a range of microarrays were produced to cover known genes in [[model organism|model]] or economically important organisms. Advances in design and manufacture of arrays improved the specificity of probes and allowed more genes to be tested on a single array. Advances in [[Fluorescence spectroscopy|fluorescence detection]] increased the sensitivity and measurement accuracy for low abundance transcripts.<ref name="#17644526" /><ref>{{cite book | first1 = Geoffrey J. | last1 = McLachlan | first2 = Kim-Anh | last2 = Do | author2-link = Kim-Anh Do | last3 = Ambroise | first3 = Christopher| name-list-style = vanc | title = Analyzing Microarray Gene Expression Data |date=2005|publisher=John Wiley & Sons|___location=Hoboken|isbn=978-0-471-72612-8}}{{page needed|date=June 2017}}</ref>

RNA-Seq is accomplished by reverse transcribing RNA ''in vitro'' and sequencing the resulting [[Complementary DNA|cDNAs]].<ref name="#19015660" /> Transcript abundance is derived from the number of counts from each transcript. The technique has therefore been heavily influenced by the development of [[DNA sequencing#High-throughput methods|high-throughput sequencing technologies]].<ref name="#23290152" /><ref name="#21191423" /> [[Massively parallel signature sequencing]] (MPSS) was an early example based on generating 16–20&nbsp;[[Base pair#Length measurements|bp]] sequences via a complex series of [[Nucleic acid hybridization|hybridisation]]s,<ref name="#10835600">{{cite journal | vauthors = Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR, Moon K, Burcham T, Pallas M, DuBridge RB, Kirchner J, Fearon K, Mao J, Corcoran K | title = Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays | journal = Nature Biotechnology | volume = 18 | issue = 6 | pages = 630–4 | date = June 2000 | pmid = 10835600 | doi = 10.1038/76469 | s2cid = 13884154 }}</ref><ref group="note">In molecular biology,&nbsp;'''hybridisation''' is a phenomenon in which single-stranded deoxyribonucleic acid ([[DNA]]) or ribonucleic acid ([[RNA]]) molecules&nbsp;[[Nucleic acid thermodynamics#Annealing|anneal]]&nbsp;to&nbsp;[[Complementarity (molecular biology)|complementary DNA or RNA]].</ref> and was used in 2004 to validate the expression of ten thousand genes in ''[[Arabidopsis thaliana]]''.<ref name="#15247925">{{cite journal | vauthors = Meyers BC, Vu TH, Tej SS, Ghazal H, Matvienko M, Agrawal V, Ning J, Haudenschild CD | title = Analysis of the transcriptional complexity of Arabidopsis thaliana by massively parallel signature sequencing | journal = Nature Biotechnology | volume = 22 | issue = 8 | pages = 1006–11 | date = August 2004 | pmid = 15247925 | doi = 10.1038/nbt992 | s2cid = 15336496 }}</ref> The earliest RNA-Seq work was published in 2006 with one hundred thousand transcripts sequenced using [[454 Life Sciences#Technology|454 technology]].<ref name="#17010196">{{cite journal | vauthors = Bainbridge MN, Warren RL, Hirst M, Romanuik T, Zeng T, Go A, Delaney A, Griffith M, Hickenbotham M, Magrini V, Mardis ER, Sadar MD, Siddiqui AS, Marra MA, Jones SJ | title = Analysis of the prostate cancer cell line LNCaP transcriptome using a sequencing-by-synthesis approach | journal = BMC Genomics | volume = 7 | pages = 246 | date = September 2006 | pmid = 17010196 | pmc = 1592491 | doi = 10.1186/1471-2164-7-246 }}</ref> This was sufficient coverage to quantify relative transcript abundance. RNA-Seq began to increase in popularity after 2008 when new [[Illumina dye sequencing|Solexa/Illumina technologies]] allowed one billion transcript sequences to be recorded.<ref name="#18599741" /><ref name="#19015660" /><ref name="#18516045">{{cite journal | vauthors = Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B | title = Mapping and quantifying mammalian transcriptomes by RNA-Seq | journal = Nature Methods | volume = 5 | issue = 7 | pages = 621–8 | date = July 2008 | pmid = 18516045 | doi = 10.1038/nmeth.1226 | s2cid = 205418589 }}</ref><ref name="#18488015">{{cite journal | vauthors = Wilhelm BT, Marguerat S, Watt S, Schubert F, Wood V, Goodhead I, Penkett CJ, Rogers J, Bähler J | title = Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution | journal = Nature | volume = 453 | issue = 7199 | pages = 1239–43 | date = June 2008 | pmid = 18488015 | doi = 10.1038/nature07002 | bibcode = 2008Natur.453.1239W | s2cid = 205213499 }}</ref> This yield now allows for the [[Quantification (science)|quantification]] and comparison of human transcriptomes.<ref>{{cite journal | vauthors = Sultan M, Schulz MH, Richard H, Magen A, Klingenhoff A, Scherf M, Seifert M, Borodina T, Soldatov A, Parkhomchuk D, Schmidt D, O'Keeffe S, Haas S, Vingron M, Lehrach H, Yaspo ML | title = A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome | journal = Science | volume = 321 | issue = 5891 | pages = 956–60 | date = August 2008 | pmid = 18599741 | doi = 10.1126/science.1160342 | bibcode = 2008Sci...321..956S | s2cid = 10013179 }}</ref>