We describe a new method for sequencing-based cross-species transcriptome comparisons and define a new metric for evaluating gene expression across species using protein-coding families as units of comparison. Using t...We describe a new method for sequencing-based cross-species transcriptome comparisons and define a new metric for evaluating gene expression across species using protein-coding families as units of comparison. Using this measure transcriptomes from different species were evaluated by mapping them to gene families and integrating the mapping results with expression data. Statistical tests were applied to the transcriptome evaluation results to identify differentially expressed families. A Perl program named Pro-Diff was compiled to im- plement this method. To evaluate the method and provide an example of its use, two liver EST transcriptomes from two closely related fish that live in different temperature zones were compared. One EST library was from a recent sequencing project of Dissosticus maw- soni, a fish that lives in cold Antarctic sea waters, while the other was newly sequenced data （available at： http：//www.fishgenome.org/ polarbank/） from Notothenia angustata, a species that lives in temperate near-shore water of southern New Zealand. Results from the com- parison were consistent with results inferred from phenotype differences and also with our previously published Gene Ontology-based method. The Pro-Diffprogram and operation manual can be downloaded from： http：//www.fishgenome.org/download/Prodiff.rar.展开更多
为了揭示猪不同肉质样品内参蛋白表达的稳定性,试验测定了北京黑猪宰后24小时pH值、肉色(L^*)值以及72小时滴水损失(drip loss,DL)值,并根据测定结果挑选出PSE(pale soft and exudative)及RFN(red firm and non-exudative)2种肉样,采用W...为了揭示猪不同肉质样品内参蛋白表达的稳定性,试验测定了北京黑猪宰后24小时pH值、肉色(L^*)值以及72小时滴水损失(drip loss,DL)值,并根据测定结果挑选出PSE(pale soft and exudative)及RFN(red firm and non-exudative)2种肉样,采用Western-blot方法检测2种肉样中3种内参蛋白β-Actin、Gapdh及β-Tubulin的表达情况。结果表明:3种内参蛋白在2种肉样中均能稳定表达,但与β-Tubulin相比,β-Actin和Gapdh的表达丰度更高。说明β-Actin、Gapdh及β-Tubulin均适合作为量化猪不同肉质样品蛋白表达的内参蛋白。展开更多
Hypo-salinity can reduce the immunological reaction in Crassostrea nippona,even lead to massive mortality.It is important to understand the molecular mechanism of oyster defense system,while quantitative real-time PCR...Hypo-salinity can reduce the immunological reaction in Crassostrea nippona,even lead to massive mortality.It is important to understand the molecular mechanism of oyster defense system,while quantitative real-time PCR can be employed in the study.However,the accuracy of quantitative real-time PCR relies on the use of suitable reference genes.In this study,the expression stability of 14 candidate reference genes including traditional housekeeping genes EF1A,TUB,TUA,GAPDH,RO21,as well as new candidate reference genes RPL5,RPL8,RPS27,RPL14,RPL4,CO3,RPS8,RPS4,CYTB in different tissues of C.nippona under salinity stress has been validated by quantitative real-time PCR.Ribosomal protein genes selected through expression analysis of transcriptome data from C.nippona generally were more stable than traditional reference genes.According to the geNorm analysis,RPL4 and RPS4 could be used as internal controls for studying gene expression in C.nippona with real-time PCR under salinity stress.展开更多
Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression ...Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rang, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rp18 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rp18 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rp18 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rp18 gene was found in all tissues examined （brain, liver, intestine, tail, testis and ovary） during R. nigromaculata development. Finally, we investigated rp18 expression under exposure to hormones. No change in rp18 mRNA expression was found under exposure to thyroid hormone （T4） and estrogen （estradiol）, whereas expression of the corresponding biomarker genes was induced. Our results show that rp18 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rp18 as a reference gene in other Rang species due to the high conservation of rp18 among the Rana genus.展开更多
基金supported by the grants from the Ministry of Science and Technology of China (No.2006AA02Z331 and 2004CB117404)the Key Project of Chinese Academy of Sciences (No.KSCX2-YW-N-020) to Liangbiao ChenNSF OPP 0636696 to C-H CC
文摘We describe a new method for sequencing-based cross-species transcriptome comparisons and define a new metric for evaluating gene expression across species using protein-coding families as units of comparison. Using this measure transcriptomes from different species were evaluated by mapping them to gene families and integrating the mapping results with expression data. Statistical tests were applied to the transcriptome evaluation results to identify differentially expressed families. A Perl program named Pro-Diff was compiled to im- plement this method. To evaluate the method and provide an example of its use, two liver EST transcriptomes from two closely related fish that live in different temperature zones were compared. One EST library was from a recent sequencing project of Dissosticus maw- soni, a fish that lives in cold Antarctic sea waters, while the other was newly sequenced data （available at： http：//www.fishgenome.org/ polarbank/） from Notothenia angustata, a species that lives in temperate near-shore water of southern New Zealand. Results from the com- parison were consistent with results inferred from phenotype differences and also with our previously published Gene Ontology-based method. The Pro-Diffprogram and operation manual can be downloaded from： http：//www.fishgenome.org/download/Prodiff.rar.
文摘为了揭示猪不同肉质样品内参蛋白表达的稳定性,试验测定了北京黑猪宰后24小时pH值、肉色(L^*)值以及72小时滴水损失(drip loss,DL)值,并根据测定结果挑选出PSE(pale soft and exudative)及RFN(red firm and non-exudative)2种肉样,采用Western-blot方法检测2种肉样中3种内参蛋白β-Actin、Gapdh及β-Tubulin的表达情况。结果表明:3种内参蛋白在2种肉样中均能稳定表达,但与β-Tubulin相比,β-Actin和Gapdh的表达丰度更高。说明β-Actin、Gapdh及β-Tubulin均适合作为量化猪不同肉质样品蛋白表达的内参蛋白。
基金This study was supported by grants from the National Natural Science Foundation of China(No.31772843)the Natural Science Foundation of Guangxi Province(No.AA17204080-4)+1 种基金the Fundamental Research Funds for the Central Universities(No.201762014)and the Ocean University of China-Auburn University Joint Research Center for Aquaculture and Environmental Science.
文摘Hypo-salinity can reduce the immunological reaction in Crassostrea nippona,even lead to massive mortality.It is important to understand the molecular mechanism of oyster defense system,while quantitative real-time PCR can be employed in the study.However,the accuracy of quantitative real-time PCR relies on the use of suitable reference genes.In this study,the expression stability of 14 candidate reference genes including traditional housekeeping genes EF1A,TUB,TUA,GAPDH,RO21,as well as new candidate reference genes RPL5,RPL8,RPS27,RPL14,RPL4,CO3,RPS8,RPS4,CYTB in different tissues of C.nippona under salinity stress has been validated by quantitative real-time PCR.Ribosomal protein genes selected through expression analysis of transcriptome data from C.nippona generally were more stable than traditional reference genes.According to the geNorm analysis,RPL4 and RPS4 could be used as internal controls for studying gene expression in C.nippona with real-time PCR under salinity stress.
基金supported by the National High Technology Research and Development Program (863) of China (No. 2012AA06A302)the Public Welfare Research Project for Environmental Protection (No. 201109048)the National Natural Science Foundation of China (No. 21077125)
文摘Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rang, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rp18 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rp18 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rp18 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rp18 gene was found in all tissues examined （brain, liver, intestine, tail, testis and ovary） during R. nigromaculata development. Finally, we investigated rp18 expression under exposure to hormones. No change in rp18 mRNA expression was found under exposure to thyroid hormone （T4） and estrogen （estradiol）, whereas expression of the corresponding biomarker genes was induced. Our results show that rp18 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rp18 as a reference gene in other Rang species due to the high conservation of rp18 among the Rana genus.
