Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue
A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene e...
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| Veröffentlicht in: | International journal of molecular sciences 2024-03, Vol.25 (5), p.2907 |
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| creator | Steinacher, Claudia Rieder, Dietmar Turner, Jasmin E Solanky, Nita Nishio, Shin-Ya Usami, Shin-Ichi Hausott, Barbara Schrott-Fischer, Anneliese Dudas, Jozsef |
| description | A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (
,
,
and
). The selected reference genes were then used to examine the effect on the expression outcome of target genes (
and
), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of
and
, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies. |
| doi_str_mv | 10.3390/ijms25052907 |
| format | Article |
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,
,
and
). The selected reference genes were then used to examine the effect on the expression outcome of target genes (
and
), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of
and
, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.</description><identifier>ISSN: 1422-0067</identifier><identifier>ISSN: 1661-6596</identifier><identifier>EISSN: 1422-0067</identifier><identifier>DOI: 10.3390/ijms25052907</identifier><identifier>PMID: 38474154</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Bones ; Gene expression ; Gene therapy ; Genes ; Genetic research ; Hearing loss ; Methods ; RNA</subject><ispartof>International journal of molecular sciences, 2024-03, Vol.25 (5), p.2907</ispartof><rights>COPYRIGHT 2024 MDPI AG</rights><rights>2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2024 by the authors. 2024</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c409t-8448f5cdadbb2b8958bf6d93fc957f6cb2c49bdb9e3f21c179cbff3db242f0e13</cites><orcidid>0000-0003-4869-4622 ; 0000-0001-7309-9820 ; 0000-0002-5935-8838 ; 0000-0002-5068-6122 ; 0000-0002-9877-3399 ; 0000-0001-8304-5543 ; 0000-0001-6640-2297</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/PMC10932218/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10932218/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27903,27904,53769,53771</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/38474154$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Steinacher, Claudia</creatorcontrib><creatorcontrib>Rieder, Dietmar</creatorcontrib><creatorcontrib>Turner, Jasmin E</creatorcontrib><creatorcontrib>Solanky, Nita</creatorcontrib><creatorcontrib>Nishio, Shin-Ya</creatorcontrib><creatorcontrib>Usami, Shin-Ichi</creatorcontrib><creatorcontrib>Hausott, Barbara</creatorcontrib><creatorcontrib>Schrott-Fischer, Anneliese</creatorcontrib><creatorcontrib>Dudas, Jozsef</creatorcontrib><title>Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue</title><title>International journal of molecular sciences</title><addtitle>Int J Mol Sci</addtitle><description>A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (
,
,
and
). The selected reference genes were then used to examine the effect on the expression outcome of target genes (
and
), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of
and
, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.</description><subject>Bones</subject><subject>Gene expression</subject><subject>Gene therapy</subject><subject>Genes</subject><subject>Genetic research</subject><subject>Hearing loss</subject><subject>Methods</subject><subject>RNA</subject><issn>1422-0067</issn><issn>1661-6596</issn><issn>1422-0067</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNpdksluFDEQhlsIREKSG2dkiQsHJnjrxSc0CpNFCkTKwtXyUp541G0PdncEb8Bj48mEaMA-uFT1_b-q5KqqtwQfMybwJ78aMq1xTQVuX1T7hFM6w7hpX-7Ee9WbnFcYU0Zr8braYx1vOan5fvX7u-q9VaOPAUWHrr_N0eLnmJR5zHyF8T7ajFSw6Gbyo9I9oGtwkCAYQGcQICMX02NUhOsEOW-EN-Nkfan5gL7AA_Rx7cMSncKoenQ-DSqgixAgoYVK6NbnPMFh9cqpPsPR03tQ3Z0ubk_OZ5dXZxcn88uZ4ViMs47zztXGKqs11Z2oO-0aK5gzom5dYzQ1XGirBTBHiSGtMNo5ZjXl1GEg7KD6vPVdT3oAayCUaXu5Tn5Q6ZeMyst_K8Hfy2V8kAQLRinpisOHJ4cUf0yQRzn4bKDvVYA4ZUlF3TQdJ0wU9P1_6CpOKZT5NlQ5pKmbQh1vqaXqQfrg4uYDyrUweBMDOF_y87ZrOGsJ5UXwcSswKeacwD23T7DcLIXcXYqCv9sd-Rn-uwXsD4TmtPI</recordid><startdate>20240302</startdate><enddate>20240302</enddate><creator>Steinacher, Claudia</creator><creator>Rieder, Dietmar</creator><creator>Turner, Jasmin E</creator><creator>Solanky, Nita</creator><creator>Nishio, Shin-Ya</creator><creator>Usami, Shin-Ichi</creator><creator>Hausott, Barbara</creator><creator>Schrott-Fischer, Anneliese</creator><creator>Dudas, Jozsef</creator><general>MDPI AG</general><general>MDPI</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-4869-4622</orcidid><orcidid>https://orcid.org/0000-0001-7309-9820</orcidid><orcidid>https://orcid.org/0000-0002-5935-8838</orcidid><orcidid>https://orcid.org/0000-0002-5068-6122</orcidid><orcidid>https://orcid.org/0000-0002-9877-3399</orcidid><orcidid>https://orcid.org/0000-0001-8304-5543</orcidid><orcidid>https://orcid.org/0000-0001-6640-2297</orcidid></search><sort><creationdate>20240302</creationdate><title>Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue</title><author>Steinacher, Claudia ; Rieder, Dietmar ; Turner, Jasmin E ; Solanky, Nita ; Nishio, Shin-Ya ; Usami, Shin-Ichi ; Hausott, Barbara ; Schrott-Fischer, Anneliese ; Dudas, Jozsef</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c409t-8448f5cdadbb2b8958bf6d93fc957f6cb2c49bdb9e3f21c179cbff3db242f0e13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><topic>Bones</topic><topic>Gene expression</topic><topic>Gene therapy</topic><topic>Genes</topic><topic>Genetic research</topic><topic>Hearing loss</topic><topic>Methods</topic><topic>RNA</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Steinacher, Claudia</creatorcontrib><creatorcontrib>Rieder, Dietmar</creatorcontrib><creatorcontrib>Turner, Jasmin E</creatorcontrib><creatorcontrib>Solanky, Nita</creatorcontrib><creatorcontrib>Nishio, Shin-Ya</creatorcontrib><creatorcontrib>Usami, Shin-Ichi</creatorcontrib><creatorcontrib>Hausott, Barbara</creatorcontrib><creatorcontrib>Schrott-Fischer, Anneliese</creatorcontrib><creatorcontrib>Dudas, Jozsef</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Research Library (Corporate)</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>International journal of molecular sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Steinacher, Claudia</au><au>Rieder, Dietmar</au><au>Turner, Jasmin E</au><au>Solanky, Nita</au><au>Nishio, Shin-Ya</au><au>Usami, Shin-Ichi</au><au>Hausott, Barbara</au><au>Schrott-Fischer, Anneliese</au><au>Dudas, Jozsef</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue</atitle><jtitle>International journal of molecular sciences</jtitle><addtitle>Int J Mol Sci</addtitle><date>2024-03-02</date><risdate>2024</risdate><volume>25</volume><issue>5</issue><spage>2907</spage><pages>2907-</pages><issn>1422-0067</issn><issn>1661-6596</issn><eissn>1422-0067</eissn><abstract>A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at -80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (
,
,
and
). The selected reference genes were then used to examine the effect on the expression outcome of target genes (
and
), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of
and
, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>38474154</pmid><doi>10.3390/ijms25052907</doi><orcidid>https://orcid.org/0000-0003-4869-4622</orcidid><orcidid>https://orcid.org/0000-0001-7309-9820</orcidid><orcidid>https://orcid.org/0000-0002-5935-8838</orcidid><orcidid>https://orcid.org/0000-0002-5068-6122</orcidid><orcidid>https://orcid.org/0000-0002-9877-3399</orcidid><orcidid>https://orcid.org/0000-0001-8304-5543</orcidid><orcidid>https://orcid.org/0000-0001-6640-2297</orcidid><oa>free_for_read</oa></addata></record> |
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| source | Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; MDPI - Multidisciplinary Digital Publishing Institute; PubMed Central |
| subjects | Bones Gene expression Gene therapy Genes Genetic research Hearing loss Methods RNA |
| title | Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue |
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