Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis)
The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference gen...
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| Veröffentlicht in: | Theriogenology 2017-04, Vol.92, p.6-13 |
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| creator | Ashish, Shende Bhure, S.K. Harikrishna, Pillai Ramteke, S.S. Muhammed Kutty, V.H. Shruthi, N. Ravi Kumar, G.V.P.P.S. Manish, Mahawar Ghosh, S.K. Mihir, Sarkar |
| description | The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.
•Assessed 13 candidate reference genes (RG) using 5 algorithms in spermatozoa.•Comprehensive ranking showed GLUT5, ATP2B4 as the two most stable RGs.•Comprehensive ranking showed B2M, MDH1as the two least stable RGs.•Expression of HSP genes was studied to evaluate RGs efficiency for normalization.•Study provides convenient RGs for normalization of gene-expression in spermatozoa. |
| doi_str_mv | 10.1016/j.theriogenology.2017.01.006 |
| format | Article |
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•Assessed 13 candidate reference genes (RG) using 5 algorithms in spermatozoa.•Comprehensive ranking showed GLUT5, ATP2B4 as the two most stable RGs.•Comprehensive ranking showed B2M, MDH1as the two least stable RGs.•Expression of HSP genes was studied to evaluate RGs efficiency for normalization.•Study provides convenient RGs for normalization of gene-expression in spermatozoa.</description><identifier>ISSN: 0093-691X</identifier><identifier>EISSN: 1879-3231</identifier><identifier>DOI: 10.1016/j.theriogenology.2017.01.006</identifier><identifier>PMID: 28237344</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; Buffalo ; Buffaloes - physiology ; Cryopreservation ; Freezing ; Gene Expression Profiling - standards ; Gene Expression Profiling - veterinary ; Gene Expression Regulation - physiology ; Male ; Normalization ; qRT-PCR ; Reference gene ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction - veterinary ; Semen Preservation - veterinary ; Sperm ; Spermatozoa - metabolism</subject><ispartof>Theriogenology, 2017-04, Vol.92, p.6-13</ispartof><rights>2017 Elsevier Inc.</rights><rights>Copyright © 2017 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-30df5bbffe894c57deabdcc4add882e41ff2ec39b2cd02dd239806f453918a9c3</citedby><cites>FETCH-LOGICAL-c386t-30df5bbffe894c57deabdcc4add882e41ff2ec39b2cd02dd239806f453918a9c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0093691X17300067$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28237344$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ashish, Shende</creatorcontrib><creatorcontrib>Bhure, S.K.</creatorcontrib><creatorcontrib>Harikrishna, Pillai</creatorcontrib><creatorcontrib>Ramteke, S.S.</creatorcontrib><creatorcontrib>Muhammed Kutty, V.H.</creatorcontrib><creatorcontrib>Shruthi, N.</creatorcontrib><creatorcontrib>Ravi Kumar, G.V.P.P.S.</creatorcontrib><creatorcontrib>Manish, Mahawar</creatorcontrib><creatorcontrib>Ghosh, S.K.</creatorcontrib><creatorcontrib>Mihir, Sarkar</creatorcontrib><title>Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis)</title><title>Theriogenology</title><addtitle>Theriogenology</addtitle><description>The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.
•Assessed 13 candidate reference genes (RG) using 5 algorithms in spermatozoa.•Comprehensive ranking showed GLUT5, ATP2B4 as the two most stable RGs.•Comprehensive ranking showed B2M, MDH1as the two least stable RGs.•Expression of HSP genes was studied to evaluate RGs efficiency for normalization.•Study provides convenient RGs for normalization of gene-expression in spermatozoa.</description><subject>Animals</subject><subject>Buffalo</subject><subject>Buffaloes - physiology</subject><subject>Cryopreservation</subject><subject>Freezing</subject><subject>Gene Expression Profiling - standards</subject><subject>Gene Expression Profiling - veterinary</subject><subject>Gene Expression Regulation - physiology</subject><subject>Male</subject><subject>Normalization</subject><subject>qRT-PCR</subject><subject>Reference gene</subject><subject>Reproducibility of Results</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - veterinary</subject><subject>Semen Preservation - veterinary</subject><subject>Sperm</subject><subject>Spermatozoa - metabolism</subject><issn>0093-691X</issn><issn>1879-3231</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkctOHDEQRa0oKExIfiHyIguy6MaPfkrZBMRjJCQ2IGVnue0y41FPe2K7Icz_8J9x0wMSu6xKrjpVt1wXoe-U5JTQ6mSdxxV46-5hcL27f8oZoXVOaE5I9QEtaFO3GWecfkQLQlqeVS39fYg-h7AmhPCqop_QIWsYr3lRLNDzUsMQrbFKRusGLAeN4UH24_x0Bnsw4GFQgJMiBGycx1Kp0cs4pzD83XoIYeIH5zeyt7u3bpMqq5epxrsdDFlcyUfQOGwhkdHtnJywxzTM4240RvYOH5-OXVohpESKNvz4gg5SIcDXfTxCdxfnt2dX2fXN5fLs13WmeFPFjBNtyq4zBpq2UGWtQXZaqUJq3TQMCmoMA8XbjilNmNaMtw2pTFHyljayVfwIHc9zt979GSFEsbFBQd_LAdwYRDouK-uqKGlCf86o8i6EdCSx9XYj_ZOgRExGibV4b5SYjBKEimRUav-2Vxq7Dei35ldnEnAxA5D--2DBi6DsZIO2HlQU2tn_U_oHsQ-zjQ</recordid><startdate>20170401</startdate><enddate>20170401</enddate><creator>Ashish, Shende</creator><creator>Bhure, S.K.</creator><creator>Harikrishna, Pillai</creator><creator>Ramteke, S.S.</creator><creator>Muhammed Kutty, V.H.</creator><creator>Shruthi, N.</creator><creator>Ravi Kumar, G.V.P.P.S.</creator><creator>Manish, Mahawar</creator><creator>Ghosh, S.K.</creator><creator>Mihir, Sarkar</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20170401</creationdate><title>Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis)</title><author>Ashish, Shende ; Bhure, S.K. ; Harikrishna, Pillai ; Ramteke, S.S. ; Muhammed Kutty, V.H. ; Shruthi, N. ; Ravi Kumar, G.V.P.P.S. ; Manish, Mahawar ; Ghosh, S.K. ; Mihir, Sarkar</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-30df5bbffe894c57deabdcc4add882e41ff2ec39b2cd02dd239806f453918a9c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Animals</topic><topic>Buffalo</topic><topic>Buffaloes - physiology</topic><topic>Cryopreservation</topic><topic>Freezing</topic><topic>Gene Expression Profiling - standards</topic><topic>Gene Expression Profiling - veterinary</topic><topic>Gene Expression Regulation - physiology</topic><topic>Male</topic><topic>Normalization</topic><topic>qRT-PCR</topic><topic>Reference gene</topic><topic>Reproducibility of Results</topic><topic>Reverse Transcriptase Polymerase Chain Reaction - veterinary</topic><topic>Semen Preservation - veterinary</topic><topic>Sperm</topic><topic>Spermatozoa - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ashish, Shende</creatorcontrib><creatorcontrib>Bhure, S.K.</creatorcontrib><creatorcontrib>Harikrishna, Pillai</creatorcontrib><creatorcontrib>Ramteke, S.S.</creatorcontrib><creatorcontrib>Muhammed Kutty, V.H.</creatorcontrib><creatorcontrib>Shruthi, N.</creatorcontrib><creatorcontrib>Ravi Kumar, G.V.P.P.S.</creatorcontrib><creatorcontrib>Manish, Mahawar</creatorcontrib><creatorcontrib>Ghosh, S.K.</creatorcontrib><creatorcontrib>Mihir, Sarkar</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Theriogenology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ashish, Shende</au><au>Bhure, S.K.</au><au>Harikrishna, Pillai</au><au>Ramteke, S.S.</au><au>Muhammed Kutty, V.H.</au><au>Shruthi, N.</au><au>Ravi Kumar, G.V.P.P.S.</au><au>Manish, Mahawar</au><au>Ghosh, S.K.</au><au>Mihir, Sarkar</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis)</atitle><jtitle>Theriogenology</jtitle><addtitle>Theriogenology</addtitle><date>2017-04-01</date><risdate>2017</risdate><volume>92</volume><spage>6</spage><epage>13</epage><pages>6-13</pages><issn>0093-691X</issn><eissn>1879-3231</eissn><abstract>The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.
•Assessed 13 candidate reference genes (RG) using 5 algorithms in spermatozoa.•Comprehensive ranking showed GLUT5, ATP2B4 as the two most stable RGs.•Comprehensive ranking showed B2M, MDH1as the two least stable RGs.•Expression of HSP genes was studied to evaluate RGs efficiency for normalization.•Study provides convenient RGs for normalization of gene-expression in spermatozoa.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>28237344</pmid><doi>10.1016/j.theriogenology.2017.01.006</doi><tpages>8</tpages></addata></record> |
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| subjects | Animals Buffalo Buffaloes - physiology Cryopreservation Freezing Gene Expression Profiling - standards Gene Expression Profiling - veterinary Gene Expression Regulation - physiology Male Normalization qRT-PCR Reference gene Reproducibility of Results Reverse Transcriptase Polymerase Chain Reaction - veterinary Semen Preservation - veterinary Sperm Spermatozoa - metabolism |
| title | Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis) |
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