{"title":"水牛精子中跨季节基因表达数据归一化参考基因的选择","authors":"Rashi Vasisth, Ankita Gurao, Meenakshi Chitkara, Gautam Kumar, Karpenahalli Ranganatha Sriranga, Manishi Mukesh, Mahesh Shivanand Dige, Pawan Singh, Rajeev Anand Kumar Aggarwal, Ranjit Singh Kataria","doi":"10.1007/s00484-024-02675-9","DOIUrl":null,"url":null,"abstract":"<p>Selection of the most stably expressed reference genes is key to monitoring accurate target gene expression across any tissue or cell type. The mRNA in spermatozoa stores valuable information related to changes in spermatogenesis due to variations in environmental conditions, especially during heat stress, which affects various sperm functions. Semen quality in buffalo bulls is significantly influenced by the seasons. In the study, a panel of nine genes was evaluated to identify the most stably expressed internal control gene (ICG) for the normalization of real-time gene expression data generated across various seasons for Murrah buffalo bulls’ spermatozoa. Sperm cells were purified from the semen samples collected during different seasons, with temperature-humidity index (THI) ranging from 80.80 ± 1.47 (hot summer) to 55.88 ± 1.98 (winter), using the BoviPure™ gradient purification method. The RNA isolated from the purified spermatozoa fraction was quality checked prior to reverse transcription and subjected to qPCR (quantitative real-time PCR) based expression analysis. An automated ‘endoGene’ pipeline was employed to apply the geNorm, NormFinder, and BestKeeper algorithms for data analysis. The result indicated that <i>GAPDH</i> and <i>PP1A</i> were the most stably expressed among the gene panel, whereas <i>ATPSF1</i> and <i>ACTB</i> were the two least stable expressed reference genes. Further, the most suitable ICGs identified were validated by normalization of real time expression data of heat stress and sperm quality genes, <i>HSFY2</i> and <i>AKAP4</i>, respectively. The genes identified would help in generating the most reliable results for the expression profiling of the genes dictating sperm quality and heat stress cope-up mechanism in buffalo spermatozoa, collected during different seasons.</p><h3 data-test=\"abstract-sub-heading\">Graphical abstract</h3>","PeriodicalId":588,"journal":{"name":"International Journal of Biometeorology","volume":null,"pages":null},"PeriodicalIF":3.0000,"publicationDate":"2024-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Selection of reference genes for normalizing gene expression data across seasons in spermatozoa of water buffalo (Bubalus bubalis)\",\"authors\":\"Rashi Vasisth, Ankita Gurao, Meenakshi Chitkara, Gautam Kumar, Karpenahalli Ranganatha Sriranga, Manishi Mukesh, Mahesh Shivanand Dige, Pawan Singh, Rajeev Anand Kumar Aggarwal, Ranjit Singh Kataria\",\"doi\":\"10.1007/s00484-024-02675-9\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Selection of the most stably expressed reference genes is key to monitoring accurate target gene expression across any tissue or cell type. The mRNA in spermatozoa stores valuable information related to changes in spermatogenesis due to variations in environmental conditions, especially during heat stress, which affects various sperm functions. Semen quality in buffalo bulls is significantly influenced by the seasons. In the study, a panel of nine genes was evaluated to identify the most stably expressed internal control gene (ICG) for the normalization of real-time gene expression data generated across various seasons for Murrah buffalo bulls’ spermatozoa. Sperm cells were purified from the semen samples collected during different seasons, with temperature-humidity index (THI) ranging from 80.80 ± 1.47 (hot summer) to 55.88 ± 1.98 (winter), using the BoviPure™ gradient purification method. The RNA isolated from the purified spermatozoa fraction was quality checked prior to reverse transcription and subjected to qPCR (quantitative real-time PCR) based expression analysis. An automated ‘endoGene’ pipeline was employed to apply the geNorm, NormFinder, and BestKeeper algorithms for data analysis. The result indicated that <i>GAPDH</i> and <i>PP1A</i> were the most stably expressed among the gene panel, whereas <i>ATPSF1</i> and <i>ACTB</i> were the two least stable expressed reference genes. Further, the most suitable ICGs identified were validated by normalization of real time expression data of heat stress and sperm quality genes, <i>HSFY2</i> and <i>AKAP4</i>, respectively. The genes identified would help in generating the most reliable results for the expression profiling of the genes dictating sperm quality and heat stress cope-up mechanism in buffalo spermatozoa, collected during different seasons.</p><h3 data-test=\\\"abstract-sub-heading\\\">Graphical abstract</h3>\",\"PeriodicalId\":588,\"journal\":{\"name\":\"International Journal of Biometeorology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2024-04-11\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"International Journal of Biometeorology\",\"FirstCategoryId\":\"89\",\"ListUrlMain\":\"https://doi.org/10.1007/s00484-024-02675-9\",\"RegionNum\":3,\"RegionCategory\":\"地球科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOPHYSICS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"International Journal of Biometeorology","FirstCategoryId":"89","ListUrlMain":"https://doi.org/10.1007/s00484-024-02675-9","RegionNum":3,"RegionCategory":"地球科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOPHYSICS","Score":null,"Total":0}
Selection of reference genes for normalizing gene expression data across seasons in spermatozoa of water buffalo (Bubalus bubalis)
Selection of the most stably expressed reference genes is key to monitoring accurate target gene expression across any tissue or cell type. The mRNA in spermatozoa stores valuable information related to changes in spermatogenesis due to variations in environmental conditions, especially during heat stress, which affects various sperm functions. Semen quality in buffalo bulls is significantly influenced by the seasons. In the study, a panel of nine genes was evaluated to identify the most stably expressed internal control gene (ICG) for the normalization of real-time gene expression data generated across various seasons for Murrah buffalo bulls’ spermatozoa. Sperm cells were purified from the semen samples collected during different seasons, with temperature-humidity index (THI) ranging from 80.80 ± 1.47 (hot summer) to 55.88 ± 1.98 (winter), using the BoviPure™ gradient purification method. The RNA isolated from the purified spermatozoa fraction was quality checked prior to reverse transcription and subjected to qPCR (quantitative real-time PCR) based expression analysis. An automated ‘endoGene’ pipeline was employed to apply the geNorm, NormFinder, and BestKeeper algorithms for data analysis. The result indicated that GAPDH and PP1A were the most stably expressed among the gene panel, whereas ATPSF1 and ACTB were the two least stable expressed reference genes. Further, the most suitable ICGs identified were validated by normalization of real time expression data of heat stress and sperm quality genes, HSFY2 and AKAP4, respectively. The genes identified would help in generating the most reliable results for the expression profiling of the genes dictating sperm quality and heat stress cope-up mechanism in buffalo spermatozoa, collected during different seasons.
期刊介绍:
The Journal publishes original research papers, review articles and short communications on studies examining the interactions between living organisms and factors of the natural and artificial atmospheric environment.
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