SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels
Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The pres...
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| creator | Grelet, Simon McShane, Ariel Hok, Eveline Tomberlin, Jensen Howe, Philip H Geslain, Renaud |
| description | Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome. |
| doi_str_mv | 10.1371/journal.pone.0177939 |
| format | Article |
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Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0177939</identifier><identifier>PMID: 28545122</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Biochemistry ; Biology and life sciences ; Copy number ; DNA microarrays ; Dynamic range ; E coli ; Embryogenesis ; Embryonic growth stage ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - growth & development ; Fractionation ; Gene expression ; Genetic aspects ; Genomes ; Hybridization ; Laboratories ; Molecular biology ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis - methods ; Orthophosphate ; Phosphates - chemistry ; Phosphorus Radioisotopes - chemistry ; Phosphorylation ; Reproducibility ; Reproducibility of Results ; Research and Analysis Methods ; Ribonucleic acid ; Ribosomes ; RNA ; RNA polymerase ; RNA, Bacterial - analysis ; RNA, Bacterial - chemistry ; RNA, Transfer - analysis ; RNA, Transfer - chemistry ; Sensitivity analysis ; Subpopulations ; Transfer RNA ; tRNA</subject><ispartof>PloS one, 2017-05, Vol.12 (5), p.e0177939-e0177939</ispartof><rights>COPYRIGHT 2017 Public Library of Science</rights><rights>2017 Grelet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Grelet et al 2017 Grelet et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-bed27c4d9b9e2525b9f34cf34e7d9f23f5271effa14c548020bbdfacbe4ff2883</citedby><cites>FETCH-LOGICAL-c692t-bed27c4d9b9e2525b9f34cf34e7d9f23f5271effa14c548020bbdfacbe4ff2883</cites><orcidid>0000-0002-5196-7540</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435355/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435355/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28545122$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Lin, Baochuan</contributor><creatorcontrib>Grelet, Simon</creatorcontrib><creatorcontrib>McShane, Ariel</creatorcontrib><creatorcontrib>Hok, Eveline</creatorcontrib><creatorcontrib>Tomberlin, Jensen</creatorcontrib><creatorcontrib>Howe, Philip H</creatorcontrib><creatorcontrib>Geslain, Renaud</creatorcontrib><title>SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. 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The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.</description><subject>Biochemistry</subject><subject>Biology and life sciences</subject><subject>Copy number</subject><subject>DNA microarrays</subject><subject>Dynamic range</subject><subject>E coli</subject><subject>Embryogenesis</subject><subject>Embryonic growth stage</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - growth & development</subject><subject>Fractionation</subject><subject>Gene expression</subject><subject>Genetic aspects</subject><subject>Genomes</subject><subject>Hybridization</subject><subject>Laboratories</subject><subject>Molecular biology</subject><subject>Nucleic Acid Hybridization</subject><subject>Oligonucleotide Array Sequence Analysis - methods</subject><subject>Orthophosphate</subject><subject>Phosphates - chemistry</subject><subject>Phosphorus Radioisotopes - chemistry</subject><subject>Phosphorylation</subject><subject>Reproducibility</subject><subject>Reproducibility of Results</subject><subject>Research and Analysis Methods</subject><subject>Ribonucleic acid</subject><subject>Ribosomes</subject><subject>RNA</subject><subject>RNA polymerase</subject><subject>RNA, Bacterial - 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| issn | 1932-6203 1932-6203 |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS) Journals Open Access; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Biochemistry Biology and life sciences Copy number DNA microarrays Dynamic range E coli Embryogenesis Embryonic growth stage Escherichia coli Escherichia coli - genetics Escherichia coli - growth & development Fractionation Gene expression Genetic aspects Genomes Hybridization Laboratories Molecular biology Nucleic Acid Hybridization Oligonucleotide Array Sequence Analysis - methods Orthophosphate Phosphates - chemistry Phosphorus Radioisotopes - chemistry Phosphorylation Reproducibility Reproducibility of Results Research and Analysis Methods Ribonucleic acid Ribosomes RNA RNA polymerase RNA, Bacterial - analysis RNA, Bacterial - chemistry RNA, Transfer - analysis RNA, Transfer - chemistry Sensitivity analysis Subpopulations Transfer RNA tRNA |
| title | SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels |
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