{"title":"为涉及小型反刍动物外周血单核细胞的基因表达研究选择可靠的参考基因","authors":"Mahanthi Vasu , Sonika Ahlawat , Vikas Choudhary , Rekha Sharma , Reena Arora , Upasna Sharma , Pooja Chhabra","doi":"10.1016/j.smallrumres.2024.107256","DOIUrl":null,"url":null,"abstract":"<div><p>Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (<em>ACTB</em>, <em>BACH1</em>, <em>B2M</em>, <em>GAPDH</em>, <em>HMBS</em>, <em>PGK1</em>, <em>PPIA</em>, <em>PPIB</em>, <em>RPLP0</em>, <em>RPL19</em>, <em>RPS9</em>, <em>RPS15</em>, <em>RPS28</em>, and <em>UXT)</em> in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that <em>PPIB, BACH1, ACTB</em>, and <em>PPIA</em> are the most suitable RGs, while <em>RPLP0, GAPDH</em> and <em>RPS15</em> are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations.</p></div>","PeriodicalId":21758,"journal":{"name":"Small Ruminant Research","volume":null,"pages":null},"PeriodicalIF":1.6000,"publicationDate":"2024-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Selection of reliable reference genes for gene expression studies involving peripheral blood mononuclear cells in small ruminants\",\"authors\":\"Mahanthi Vasu , Sonika Ahlawat , Vikas Choudhary , Rekha Sharma , Reena Arora , Upasna Sharma , Pooja Chhabra\",\"doi\":\"10.1016/j.smallrumres.2024.107256\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (<em>ACTB</em>, <em>BACH1</em>, <em>B2M</em>, <em>GAPDH</em>, <em>HMBS</em>, <em>PGK1</em>, <em>PPIA</em>, <em>PPIB</em>, <em>RPLP0</em>, <em>RPL19</em>, <em>RPS9</em>, <em>RPS15</em>, <em>RPS28</em>, and <em>UXT)</em> in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that <em>PPIB, BACH1, ACTB</em>, and <em>PPIA</em> are the most suitable RGs, while <em>RPLP0, GAPDH</em> and <em>RPS15</em> are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations.</p></div>\",\"PeriodicalId\":21758,\"journal\":{\"name\":\"Small Ruminant Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2024-03-13\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Small Ruminant Research\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0921448824000622\",\"RegionNum\":3,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"AGRICULTURE, DAIRY & ANIMAL SCIENCE\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Small Ruminant Research","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0921448824000622","RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"AGRICULTURE, DAIRY & ANIMAL SCIENCE","Score":null,"Total":0}
Selection of reliable reference genes for gene expression studies involving peripheral blood mononuclear cells in small ruminants
Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, and UXT) in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that PPIB, BACH1, ACTB, and PPIA are the most suitable RGs, while RPLP0, GAPDH and RPS15 are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations.
期刊介绍:
Small Ruminant Research publishes original, basic and applied research articles, technical notes, and review articles on research relating to goats, sheep, deer, the New World camelids llama, alpaca, vicuna and guanaco, and the Old World camels.
Topics covered include nutrition, physiology, anatomy, genetics, microbiology, ethology, product technology, socio-economics, management, sustainability and environment, veterinary medicine and husbandry engineering.