# Cap analysis of gene expression > Mediated Wiki article. Canonical URL: https://mediated.wiki/source/Cap_analysis_of_gene_expression > Markdown URL: https://mediated.wiki/source/Cap_analysis_of_gene_expression.md > Source: https://en.wikipedia.org/wiki/Cap_analysis_of_gene_expression > Source revision: 1324478100 > License: Creative Commons Attribution-ShareAlike 4.0 International (https://creativecommons.org/licenses/by-sa/4.0/) '''Cap analysis of gene expression''' ('''CAGE''') is a [gene expression](/source/gene_expression) technique used in molecular biology to produce a snapshot of the [5′ end](/source/Directionality_(molecular_biology)) of the [messenger RNA](/source/messenger_RNA) population in a biological sample (the [transcriptome](/source/transcriptome)). The small fragments (historically 27 [nucleotide](/source/nucleotide)s long, but now limited only by sequencing technologies) from the very beginnings of mRNAs (5' ends of [capped](/source/5'_cap) transcripts) are extracted, [reverse-transcribed](/source/reverse_transcription) to cDNA, [PCR](/source/Polymerase_chain_reaction) amplified (if needed) and [sequenced](/source/DNA_sequencing). CAGE was first published by Hayashizaki, [Carninci](/source/Piero_Carninci) and co-workers in 2003.{{cite journal | journal=Proc Natl Acad Sci U S A | date=2003-12-23 | volume=100 | issue=26 | pages=15776–81 | pmid=14663149 | doi=10.1073/pnas.2136655100 | title=Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage | pmc=307644 | last1 = Shiraki | first1 = T | last2 = Kondo | first2 = S | last3 = Katayama | first3 = S | bibcode=2003PNAS..10015776S |display-authors=et al| doi-access=free }} CAGE has been extensively used within the [FANTOM](/source/FANTOM) research projects. ==Analysis== The output of CAGE is a set of short nucleotide sequences (often called ''tags'' in analogy to [expressed sequence tag](/source/expressed_sequence_tag)s) with their observed counts. Copy numbers of CAGE tags provide a digital quantification of the RNA transcript abundances in biological samples. Using a reference genome, a researcher can usually determine, with some confidence, the original [mRNA](/source/mRNA) (and therefore which [gene](/source/gene)) the tag was extracted from. Unlike a similar technique [serial analysis of gene expression](/source/serial_analysis_of_gene_expression) (SAGE) in which tags come from other parts of transcripts, CAGE is primarily used to locate exact [transcription](/source/transcription_(genetics)) start sites in the genome. This knowledge in turn allows a researcher to investigate [promoter](/source/promoter_(biology)) structure necessary for gene expression. CAGE tags tend to start with an extra [guanine](/source/guanine) (G) that is not encoded in the genome, which is attributed to the template-free 5′-extension during the first-strand [cDNA](/source/cDNA) synthesis{{cite journal |last1= Zhao |first1= Xiaobei |year= 2011 |title= Systematic Clustering of Transcription Start Site Landscapes |journal= PLOS ONE |volume= 6 |issue= 8 |article-number= e23409 |doi= 10.1371/journal.pone.0023409 |pmid=21887249 |pmc=3160847 |bibcode= 2011PLoSO...623409Z |doi-access= free }} or reverse-transcription of the cap itself.{{cite journal |last1=Ohtake |first1=Hideki |last2=Ohkoto |first2=Kuniyo |last3=Ishimaru |first3=Yoshihiro |last4=Kato |first4=Seishi |year=2004 |title = Determination of the capped site sequence of mRNA based on the detection of cap-dependent nucleotide addition using an anchor ligation method |journal=DNA Res. |volume=11 |issue=4 |pages=305–9 |pmid=15500255 |doi=10.1093/dnares/11.4.305|doi-access=free }} When not corrected, this can induce erroneous mapping of CAGE tags, for instance to nontranscribed pseudogenes. On the other hand, this addition of Gs was also utilised as a signal to filter more reliable TSS peaks.{{cite journal |last1= Cumbie |first1= Jason |year= 2015 |title= NanoCAGE-XL and CapFilter: an approach to genome wide identification of high confidence transcription start sites |journal= BMC Genomics |volume= 16 |issue= 1 |article-number= 597 |doi= 10.1186/s12864-015-1670-6 |pmid= 26268438 |pmc= 4534009 |doi-access= free }} ==History== The original CAGE method (Shiraki ''et al.'', 2003) was using CAP Trapper{{cite journal |last1= Carninci |first1= Piero |year= 1996 |title= High-efficiency full-length cDNA cloning by biotinylated CAP trapper. |journal= Genomics |volume= 37 |issue= 3 |pages= 327–36 |pmid= 8938445 |doi=10.1006/geno.1996.0567 }} for capturing the 5′ ends, oligo-dT primers for synthesizing the cDNAs, the [type IIs restriction enzyme](/source/Restriction_enzyme) MmeI for cleaving the tags, and the [Sanger method](/source/Sanger_sequencing) for sequencing them. Random reverse-transcription primers were introduced in 2006 by Kodzius ''et al.''{{cite journal |last1= Kodzius |first1= Rimantas |year= 2006 |title= CAGE: cap analysis of gene expression. |journal= Nat Methods |volume= 3 |issue= 3 |pages= 211–22 |doi= 10.1038/nmeth0306-211 |pmid= 16489339 |s2cid= 32641130 }} to better detect the non-polyadenylated RNAs. In ''DeepCAGE'' (Valen ''et al.'', 2008),{{cite journal |last1= Valen |first1= Eivind |year= 2009 |title= Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE. |journal= Genome Res. |volume= 19 |issue= 2 |pages= 255–265 |doi= 10.1101/gr.084541.108 |pmid= 19074369 |pmc=2652207 }} the tag concatemers were sequenced at a higher throughput on the [454](/source/454_Life_Sciences) "''next-generation''" sequencing platform. In 2008, barcode multiplexing was added to the DeepCAGE protocol (Maeda ''et al.'', 2008).{{cite journal |last1= Maeda |first1= Norihiro |year= 2008 |title= Development of a DNA barcode tagging method for monitoring dynamic changes in gene expression by using an ultra high-throughput sequencer. |journal= BioTechniques |volume= 45 |issue= 1 |pages= 95–7 |doi= 10.2144/000112814 |pmid= 18611171 |url= http://www.biotechniques.com/article/000112814 |access-date= 2016-04-28 |doi-access= free }} In ''nanoCAGE'' (Plessy ''et al.'', 2010),{{cite journal |last1= Plessy |first1= Charles |year= 2010 |title= Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan |journal= Nat Methods |volume= 7 |issue= 7 |pages= 528–34 |doi= 10.1038/nmeth.1470 |pmid= 20543846 |pmc=2906222 }} the 5′ ends or RNAs were captured with the template-switching method instead of CAP Trapper, in order to analyze smaller starting amounts of total RNA. Longer tags were cleaved with the [type III restriction enzyme](/source/Restriction_enzyme) EcoP15I and directly sequenced on the [Solexa (then Illumina)](/source/DNA_sequencing) platform without concatenation. The ''CAGEscan'' methodology (Plessy ''et al.'', 2010), where the enzymatic tag cleavage is skipped, and the 5′ cDNAs sequenced [paired-end](/source/Paired-end_tag), was introduced in the same article to connect novel promoters to known annotations. With ''HeliScopeCAGE'' (Kanamori-Katayama ''et al.'', 2011),{{cite journal |last1= Kanamori-Katayama |first1= Mutsumi |year= 2011 |title= Unamplified cap analysis of gene expression on a single-molecule sequencer. |journal= Genome Res. |volume= 21 |issue= 7 |pages= 1150–9 |doi= 10.1101/gr.115469.110 |pmid= 21596820 |pmc=3129257 }} the CAP-trapped CAGE protocol was changed to skip the enzymatic tag cleavage and sequence directly the capped 5′ ends on the [HeliScope](/source/Helicos_single_molecule_fluorescent_sequencing) platform, without PCR amplification. It was then automated by Itoh ''et al.''{{cite journal |last1= Itoh |first1= Masayoshi |year= 2012 |title= Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer. |journal= PLOS ONE |volume= 7 |issue= 1 |article-number= e30809 |doi= 10.1371/journal.pone.0030809 |pmid= 22303458 |pmc=3268765 |bibcode= 2012PLoSO...730809I |doi-access= free }} in 2012. In 2012, the standard CAGE protocol was updated by Takahashi ''et al.''{{cite journal |last1= Takahashi |first1= Hazuki |year= 2012 |title= 5' end-centered expression profiling using cap-analysis gene expression and next-generation sequencing. |journal= Nat Protoc |volume= 7 |issue= 3 |pages= 542–61 |doi= 10.1038/nprot.2012.005 |pmid= 22362160 |pmc=4094379 }} to cleave tags with EcoP15I and sequence them on the Illumina-Solexa platform. In 2013, Batut ''et al.''{{cite journal |last1= Batut |first1= Philippe |year= 2013 |title= High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression. |journal= Genome Res. |volume= 23 |issue= 1 |pages= 169–80 |doi= 10.1101/gr.139618.112 |pmid= 22936248 |pmc=3530677 }} combined CAP trapper, template switching, and 5′-phosphate-dependent exonuclease digestion in ''RAMPAGE'' to maximize promoter specificity. In 2014, Murata ''et al.''{{cite book |last1=Murata |first1=Mitsuyoshi |title=Transcription Factor Regulatory Networks |year=2014 |chapter=Detecting Expressed Genes Using CAGE |volume=1164 |pages=67–85 |doi=10.1007/978-1-4939-0805-9_7 |pmid=24927836 |series=Methods in Molecular Biology |isbn=978-1-4939-0804-2 }} published the ''nAnTi-CAGE'' protocol, where capped 5′ ends are sequenced on the Illumina platform with no PCR amplification and no tag cleavage. In 2017, Poulain ''et al.''{{cite book |last1=Poulain |first1=Stéphane |chapter=NanoCAGE: A Method for the Analysis of Coding and Noncoding 5′-Capped Transcriptomes |year=2017 |title=Promoter Associated RNA |volume=1543 |pages=57–109 |doi=10.1007/978-1-4939-6716-2_4 |pmid=28349422 |series=Methods in Molecular Biology |isbn=978-1-4939-6714-8 }} updated the ''nanoCAGE'' protocol to use the ''tagmentation'' method (based on [Tn5 transposition](/source/Transposase)) for multiplexing. In 2018, Cvetesic '' et al.''{{cite journal |last1=Cvetesic |first1=Nevena |year=2018 |title=SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA |journal=Genome Research |volume=28 |issue=12 |pages=1943–1956 |doi=10.1101/gr.235937.118 |pmid=30404778|pmc=6280763 }} increased the sensitivity of CAP-trapped CAGE by introducing selectively degradable carrier RNA (SLIC-CAGE, "Super-Low Input Carrier-CAGE"). In 2021, Takahashi ''et al.''{{cite book |last1=Takahashi |first1=Hazuki |title=Enhancers and Promoters |year=2021 |chapter= Low Quantity Single Strand CAGE (LQ-ssCAGE) Maps Regulatory Enhancers and Promoters |series=Methods Mol Biol. |volume=2351 |pages=67–90 |doi=10.1007/978-1-0716-1597-3_4 |pmid=34382184 |isbn=978-1-0716-1596-6 |s2cid=243553132 }} simplified the sequencing of CAGE libraries on Illumina sequencers by skipping second-strand synthesis directly loading single-strand cDNAs (Low Quantity Single Strand CAGE, "LQ-ssCAGE"). In 2025, Delobel ''et al.''{{Cite journal |last=Delobel |first=Diane |last2=Nishiyori-Sueki |first2=Hiromi |last3=Nisoli |first3=Ilaria |last4=Kawaji |first4=Hideya |last5=Robbe |first5=Pauline |last6=Carninci |first6=Piero |last7=Takahashi |first7=Hazuki |date=2025-03-21 |title=Protocol for direct cDNA cap analysis of gene expression for paired-end patterned flow cell sequencing |journal=STAR protocols |volume=6 |issue=1 |article-number=103594 |doi=10.1016/j.xpro.2024.103594 |issn=2666-1667 |pmc=11851279 |pmid=39921863}} updated the ''nAnTi-CAGE'' protocol to introduce unique dual indexing, to mitigate the index hopping artifacts that are more frequent in the patterned flow cells used in the 2-color Illumina instruments. ==See also== *[Serial analysis of gene expression](/source/Serial_analysis_of_gene_expression) *[RNA-Seq](/source/RNA-Seq) *[Transcriptomics](/source/Transcriptomics) ==References== {{reflist|35em}} ==External links== * [https://web.archive.org/web/20110301224715/http://www.osc.riken.jp/english/activity/cage/ CAGE homepage] at the [RIKEN](/source/RIKEN) Omics Science Center. * [http://fantom.gsc.riken.jp/protocols/ Protocols page] on the FANTOM5 website. {{DEFAULTSORT:Cap Analysis Gene Expression}} Category:Molecular biology Category:Gene expression --- Adapted from the Wikipedia article [Cap analysis of gene expression](https://en.wikipedia.org/wiki/Cap_analysis_of_gene_expression) by Wikipedia contributors ([contributor history](https://en.wikipedia.org/wiki/Cap_analysis_of_gene_expression?action=history)). 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