AIM:Normalizing the results of real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)is essential for the accuracy of analysis.Commonly used approaches include input nucleic acid standardizat...AIM:Normalizing the results of real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)is essential for the accuracy of analysis.Commonly used approaches include input nucleic acid standardization(ΔCt method),normalization against a single internal reference gene(ΔΔCt method),and geometric averaging of multiple reference gene abundance using statistical software.We evaluated these approaches in the liver of db/db mice,a typical model of fatty liver disease.METHODS:Seven reference genes,β-actin(ACTB),eukaryotic initiation factor(e IF)5,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),hydroxymethylbilane synthase(HMBS),hypoxanthineguanine phosphoribosyltransferase(HPRT)1,polymerase(RNA)II(DNA directed)polypeptide A(Polr2A)and ribosomal protein P〈0(RPLP〈0),were evaluated using software of ge Norm and Norm Finder.Hepatic lipogenesis genes,such as thyroid hormone-responsive protein(Thrsp),stearoyl-Co A desaturase(SCD)1,sterol regulatory element-binding protein(SREBP)1c and fatty acid synthase(FAS),were used as target genes of interest.RESULTS:The expression levels of all target genes and GAPDH were significantly elevated(P〈0.05)in db/db mouse livers by theΔCt method.ACTB and HMBS were the most stable genes calculated by the software of ge Norm.Norm Finder analysis indicated that ACTB was the most stable gene,and the best combination of 2 genes was GAPDH and RPLP〈0.Normalization against a single internal reference gene of ACTB or RPLP〈0,the geometric mean of ACTB and HMBS,or GAPDH and RPLP〈0 showed similar results that the expression levels of Thrsp,SCD1 and FAS,but not SREBP1 c increased(P〈0.05)in the liver of db/db mice.CONCLUSION:TheΔCt approach ensures a meaningful and biologically significant appraisal of gene expression.Use of the software like ge Norm or Norm Finder should be integrated withΔCt method.展开更多
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
基金Supported by National Natural Science Foundation of China(No.81370477No.81370918)+2 种基金Science and Technology Research Outstanding Youth Fund of Hebei College(No.Y2011117)Undergraduate Innovation Project(No.201410081061No.X2014026)
文摘AIM:Normalizing the results of real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)is essential for the accuracy of analysis.Commonly used approaches include input nucleic acid standardization(ΔCt method),normalization against a single internal reference gene(ΔΔCt method),and geometric averaging of multiple reference gene abundance using statistical software.We evaluated these approaches in the liver of db/db mice,a typical model of fatty liver disease.METHODS:Seven reference genes,β-actin(ACTB),eukaryotic initiation factor(e IF)5,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),hydroxymethylbilane synthase(HMBS),hypoxanthineguanine phosphoribosyltransferase(HPRT)1,polymerase(RNA)II(DNA directed)polypeptide A(Polr2A)and ribosomal protein P〈0(RPLP〈0),were evaluated using software of ge Norm and Norm Finder.Hepatic lipogenesis genes,such as thyroid hormone-responsive protein(Thrsp),stearoyl-Co A desaturase(SCD)1,sterol regulatory element-binding protein(SREBP)1c and fatty acid synthase(FAS),were used as target genes of interest.RESULTS:The expression levels of all target genes and GAPDH were significantly elevated(P〈0.05)in db/db mouse livers by theΔCt method.ACTB and HMBS were the most stable genes calculated by the software of ge Norm.Norm Finder analysis indicated that ACTB was the most stable gene,and the best combination of 2 genes was GAPDH and RPLP〈0.Normalization against a single internal reference gene of ACTB or RPLP〈0,the geometric mean of ACTB and HMBS,or GAPDH and RPLP〈0 showed similar results that the expression levels of Thrsp,SCD1 and FAS,but not SREBP1 c increased(P〈0.05)in the liver of db/db mice.CONCLUSION:TheΔCt approach ensures a meaningful and biologically significant appraisal of gene expression.Use of the software like ge Norm or Norm Finder should be integrated withΔCt method.
基金The National Natural Science Foundation of China-Shandong Joint Fund for Marine Science Research Centers under contract No.U1406403the State Key Program of National Natural Science of China under contract No.61533011China Postdoctoral Science Foundation under contract Nos 2014M551969 and 2015T80754
文摘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.
