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Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time PCR 被引量:5
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作者 LI Bing CHEN Changsheng +2 位作者 XU Yan JI Dehua XIE Chaotian 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2014年第9期152-159,共8页
Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these char... Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes (18S rRNA (18S), ubiquitin-conju-ating enzyme (UBC), actin (ACT), β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phos- phate dehydrogenase (GAPDH) examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (Ct) method and by two different software packages: geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses. 展开更多
关键词 Pyropia haitanensis quantitative real-time PCR internal control genes gene expression
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Selection of suitable internal controls for gene expression normalization in rats with spinal cord injury 被引量:3
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作者 Wei Liu Jie Yu +2 位作者 Yi-Fan Wang Qian-Qian Shan Ya-Xian Wang 《Neural Regeneration Research》 SCIE CAS CSCD 2022年第6期1387-1392,共6页
There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research.In ... There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research.In this study,we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes:Actb,Ankrd27,CypA,Gapdh,Hprt1,Mrpl10,Pgk1,Rictor,Rn18s,Tbp,Ubc,Ubxn11,and Ywhaz.Our results show that the expression of Ankrd27,Ubc,and Tbp were stable after spinal cord injury,while Actb was the most unstable internal control gene.Ankrd27,Ubc,Tbp,and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression.Target gene expression levels and changes over time were similar when Ankrd27,Ubc,and Tbp were used as internal controls but different when Actb was used as an internal control.We recommend that Ankrd27,Ubc,and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research.This study was approved by the Administration Committee of Experimental Animals,Jiangsu Province,China(approval No.20180304-008)on March 4,2018. 展开更多
关键词 geNorm analysis reference genes internal control genes NORMALIZATION NormFinder analysis reverse transcription-quantitative polymerase chain reaction spinal cord injury stability of gene expression
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Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis 被引量:1
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作者 王霞 冯建华 +3 位作者 黄爱优 何林文 牛建峰 王广策 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2017年第6期1432-1441,共10页
Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the... Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis. 展开更多
关键词 real-time quantitative PCR housekeeping genes internal control genes stress responding Pyropia haitanensis
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Identification of stable internal control genes for accurate normalization of real-time quantitative PCR data in testicular tissue from two breeds of cattle
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作者 Pradeep Nag Ankur Sharma +6 位作者 Elango Kamaraj Arumugam Kumaresan Tirtha Kumar Datta Ayyasamy Manimaran Nilendu Paul Sakthivel Jeyakumar Kerekoppa P.Ramesha 《Asian pacific Journal of Reproduction》 2020年第5期247-255,共9页
Objective:To assess the stability of 10 candidate internal control genes(ICGs),namely GAPDH,ACTB,RPL23,RPS15A,ATPSF1,GLUT5,HMBS,ATP2B4,PPIA,and BRP to normalize the transcriptional data from testes samples of Zebu and... Objective:To assess the stability of 10 candidate internal control genes(ICGs),namely GAPDH,ACTB,RPL23,RPS15A,ATPSF1,GLUT5,HMBS,ATP2B4,PPIA,and BRP to normalize the transcriptional data from testes samples of Zebu and crossbred bulls.Methods:Total RNA was isolated from testicular tissue of Zebu and crossbred bulls(n=6 each)between 2-8 years of age.cDNA was synthesized,and the quantitative real-time polymerase chain reaction(PCR)was performed.The cycle threshold values were used for the analysis of the stability of ICGs.Four different statistical algorithms:geNorm,Normfinder,BestKeeper,and RefFinder,were used to assess the stability of these genes.Results:ATPSF1,HMBS,PPIA,and RPS15A were the most reliable and stable ICGs for Zebu testes,and ATPSF1,RPL23,and PPIA for crossbred testes.Conclusions:A panel of stable ICGs(ATPSF1,HMBS,PPIA,RPS15A for Zebu and ATPSF1,RPL23,and PPIA for crossbred)for normalization of gene expression data in testes samples can be helpful for researchers to conduct functional genomics studies at the testicular level in cattle bulls. 展开更多
关键词 Cattle bulls gene expression internal control genes NORMALIZATION qPCR Testis
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Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata 被引量:2
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作者 濮菲 杨丙晔 柯才焕 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2015年第4期838-845,共8页
Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and vali... Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 (ACT-2), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), β-tubulin (β-TUB), and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene (Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve. 展开更多
关键词 Crassostrea angulata gene expression quantitative real-time PCR internal control gene G protein β-subunit gene
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Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR 被引量:2
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作者 DENG Yunyan HU Zhangxi +1 位作者 MA Zhaopeng TANG Ying Zhong 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2016年第8期106-112,共7页
The accurate measurement of gene expression via quantitative real-time reverse transcription PCR(q RT-PCR)heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant... The accurate measurement of gene expression via quantitative real-time reverse transcription PCR(q RT-PCR)heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA(18S), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), α-tubulin(TUA), β-tubulin(TUB), actin(ACT) and cytochrome oxidase subunit 1(COX1), were evaluated for their expression stability with q RT-PCR and three statistical algorithms(Ge Norm, Norm Finder, and Best Keeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages(resting cysts), different growth stages, in darkness, exposed to abscisic acid(ABA) and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates regarding quantification of gene expression at different experimental scenarios, which will facilitate more accurate and widespread use of q RT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species. 展开更多
关键词 Akashiwo sanguinea dinoflagellate internal control qRT-PCR resting cyst reference gene
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