Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves
Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not su...
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| Veröffentlicht in: | PloS one 2014-08, Vol.9 (8), p.e105601-e105601 |
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| creator | Gambarotta, Giovanna Ronchi, Giulia Friard, Olivier Galletta, Pantaleo Perroteau, Isabelle Geuna, Stefano |
| description | Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs. |
| doi_str_mv | 10.1371/journal.pone.0105601 |
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Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0105601</identifier><identifier>PMID: 25144298</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Algorithms ; Analysis ; Animals ; Biology and life sciences ; Degeneration ; Disease Models, Animal ; Female ; Gene expression ; Gene Expression Profiling ; Genes ; Genes, Essential ; Identification ; Injuries ; Injury analysis ; Medicine and Health Sciences ; Myelination ; Nerves ; Nervous system ; Neurosciences ; Normalizing ; Organs ; Peripheral Nerve Injuries - diagnosis ; Peripheral Nerve Injuries - genetics ; Peripheral nerves ; Pseudogenes ; Rats ; Real time ; Real-Time Polymerase Chain Reaction ; Regeneration ; Reproducibility of Results ; Research and Analysis Methods ; Ribonucleic acid ; RNA ; RNA Stability ; Tissues</subject><ispartof>PloS one, 2014-08, Vol.9 (8), p.e105601-e105601</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Gambarotta 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>2014 Gambarotta et al 2014 Gambarotta et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-f3a7976fabec89ff736d28b542a7b91b0ab38b1e5a5e258f98ce38175acae6303</citedby><cites>FETCH-LOGICAL-c692t-f3a7976fabec89ff736d28b542a7b91b0ab38b1e5a5e258f98ce38175acae6303</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140797/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140797/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79472,79473</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25144298$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Price, Theodore John</contributor><creatorcontrib>Gambarotta, Giovanna</creatorcontrib><creatorcontrib>Ronchi, Giulia</creatorcontrib><creatorcontrib>Friard, Olivier</creatorcontrib><creatorcontrib>Galletta, Pantaleo</creatorcontrib><creatorcontrib>Perroteau, Isabelle</creatorcontrib><creatorcontrib>Geuna, Stefano</creatorcontrib><title>Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.</description><subject>Algorithms</subject><subject>Analysis</subject><subject>Animals</subject><subject>Biology and life sciences</subject><subject>Degeneration</subject><subject>Disease Models, Animal</subject><subject>Female</subject><subject>Gene expression</subject><subject>Gene Expression Profiling</subject><subject>Genes</subject><subject>Genes, Essential</subject><subject>Identification</subject><subject>Injuries</subject><subject>Injury analysis</subject><subject>Medicine and Health Sciences</subject><subject>Myelination</subject><subject>Nerves</subject><subject>Nervous system</subject><subject>Neurosciences</subject><subject>Normalizing</subject><subject>Organs</subject><subject>Peripheral Nerve Injuries - diagnosis</subject><subject>Peripheral Nerve Injuries - 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John</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-08-21</date><risdate>2014</risdate><volume>9</volume><issue>8</issue><spage>e105601</spage><epage>e105601</epage><pages>e105601-e105601</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25144298</pmid><doi>10.1371/journal.pone.0105601</doi><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Algorithms Analysis Animals Biology and life sciences Degeneration Disease Models, Animal Female Gene expression Gene Expression Profiling Genes Genes, Essential Identification Injuries Injury analysis Medicine and Health Sciences Myelination Nerves Nervous system Neurosciences Normalizing Organs Peripheral Nerve Injuries - diagnosis Peripheral Nerve Injuries - genetics Peripheral nerves Pseudogenes Rats Real time Real-Time Polymerase Chain Reaction Regeneration Reproducibility of Results Research and Analysis Methods Ribonucleic acid RNA RNA Stability Tissues |
| title | Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves |
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