Quantitative real-time PCR(qPCR)is widely used for gene expression analysis,but its accuracy critically depends on stable internal reference genes for normalization.In marine invertebrates,especially non-model taxa su...Quantitative real-time PCR(qPCR)is widely used for gene expression analysis,but its accuracy critically depends on stable internal reference genes for normalization.In marine invertebrates,especially non-model taxa such as cephalopods,systematic evaluation of reference genes is limited,leading to potential bias.The cuttlefish Sepiella japonica is ecologically and economically important in China,yet previous molecular studies have often relied on single unvalidated reference genes,which may compromise data reliability.This study aimed to systematically evaluate the stability of five commonly used reference genes(18S,ef-1α,ef-1γ,gapdh,andβ-actin)across multiple tissues and sexes of S.japonica,and to identify the most suitable reference genes and optimal number for qPCR normalization.Fifteen to sixteen tissue types were collected from ten healthy adults(five males and five females).Total RNA was extracted,reverse-transcribed,and analyzed by qPCR.Gene stability was assessed using four algorithms(geNorm,NormFinder,BestKeeper,andΔCt)integrated with RefFinder,and the optimal gene number was determined using geNorm pairwise variation(V_(n/n+1)<0.15).Four transcriptome-derived genes(creld2,cd109,acy1,and miox)were used for validation.The C_(t)values of the five genes ranged from 15.47 to 20.83.β-actin and gapdh showed pronounced variability in expression stability among tissues and sexes,indicating their limited suitability for normalization.18S exhibited the highest expression(mean C_(t):15.47-16.29)and lowest variability but displayed sex-biased expression,whereas ef-1αand ef-1γremained consistently stable across most tissues in both sexes,with ef-1αbeing the most robust and showing no sex-related bias.Although specific rankings varied among tissues and sexes,the comprehensive results indicated that ef-1αand ef-1γpossessed the highest overall stability,followed by 18S,whileβ-actin and gapdh were the least stable.The final comprehensive rankings were ef-1γ>ef-1α>18S>gapdh>β-actin(male)and ef-1α>ef-1γ>18S>gapdh>β-actin(female).geNorm analysis(V2/3<0.15)indicated that two genes,mainly ef-1αand ef-1γ,were generally sufficient for reliable normalization in most tissues.Validation confirmed that normalization using the stable ef-1αand ef-1γaccurately reflected the expression differences among tissues,whereasβ-actin and gapdh can bias or confound statistical analyses.ef-1αand ef-1γare identified as the most reliable reference gene combination for qPCR analysis in S.japonica,while 18S can serve as an auxiliary gene for within-sex comparisons.The use ofβ-actin or gapdh alone is not recommended.This study establishes a systematic framework for selecting reliable reference genes in S.japonica,thereby facilitating robust qPCR normalization and providing a foundation for future gene expression research in S.japonica and other cephalopods.展开更多
脂多糖(LPS)诱导的免疫应激下,选择适于校正肉鸡肝脏、心脏和回肠组织中目的基因表达量的内参基因。将30只1日龄的肉鸡随机分为对照组与处理组,21日龄时,处理组注射1 mg/kg的LPS,对照组注射等量灭菌生理盐水,2 h后,每组各取7只肉鸡的肝...脂多糖(LPS)诱导的免疫应激下,选择适于校正肉鸡肝脏、心脏和回肠组织中目的基因表达量的内参基因。将30只1日龄的肉鸡随机分为对照组与处理组,21日龄时,处理组注射1 mg/kg的LPS,对照组注射等量灭菌生理盐水,2 h后,每组各取7只肉鸡的肝脏、心脏和回肠组织。通过实时荧光定量PCR技术(RT-q PCR)对各组织中β-肌动蛋白(ACTB)、β2-微球蛋白(B2M)、18 S核糖体RNA(18 S r RNA)、28 S核糖体RNA(28 S r RNA)和3-磷酸甘油醛脱氢酶(GAPDH)共5个内参基因的表达定量,利用Ref Finder和Ge Norm软件分析内参基因的稳定性及配对差异。结果显示:LPS刺激下,5个内参基因的表达水平有不同程度的变化。18 S r RNA、ACTB和GAPDH适于肝脏中目的基因表达量的校正;B2M和GAPDH适于心脏中目的基因表达量的校正;18 S r RNA和ACTB适于回肠中目的基因表达量的校正。选择表达稳定性高的内参基因适于校正目的基因的表达量,RT-q PCR应根据不同的研究对象进行选择,建议选择2个或2个以上的内参基因。展开更多
BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,...BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.展开更多
Quantitative real-time PCR(qRT-PCR)is widely used for investigating gene expression patterns and has many advantages,including its high sensitivity,fidelity,and specificity.Selecting a satisfactory internal reference ...Quantitative real-time PCR(qRT-PCR)is widely used for investigating gene expression patterns and has many advantages,including its high sensitivity,fidelity,and specificity.Selecting a satisfactory internal reference gene is crucial for obtaining precise gene expression results in qRT-PCR analyses.In this study,the transcriptomic data of 2 potato varieties were screened for housekeeping genes with stable expression patterns.A total of 77 putative genes were selected,which were highly and stably expressed.Then,qRT-PCR analyses were performed to examine the expression levels of these 77 candidate reference genes in various potato tissues,including leaves,flowers,stolons,and tubers.Gene expression was represented by analyzing the Ct values at given threshold.Through geNorm and NormFinder program analyses,10 candidate genes with the most stable expression patterns were obtained,including RPL19,RPS15,RPS9,EF1α,TrxP1,RPS8,NTF,CAM,AACM,and RPS28.Moreover,through the comprehensive analyses of 4 statistical algorithms(i.e.,geNorm,NormFinder,BestKeeper,and RefFinder),results indicated that the most appropriate internal reference genes were RPL19 and EF1α.The obtained stable reference genes will contribute to future qRT-PCR analyses on potato tissue-related gene expression.展开更多
文摘Quantitative real-time PCR(qPCR)is widely used for gene expression analysis,but its accuracy critically depends on stable internal reference genes for normalization.In marine invertebrates,especially non-model taxa such as cephalopods,systematic evaluation of reference genes is limited,leading to potential bias.The cuttlefish Sepiella japonica is ecologically and economically important in China,yet previous molecular studies have often relied on single unvalidated reference genes,which may compromise data reliability.This study aimed to systematically evaluate the stability of five commonly used reference genes(18S,ef-1α,ef-1γ,gapdh,andβ-actin)across multiple tissues and sexes of S.japonica,and to identify the most suitable reference genes and optimal number for qPCR normalization.Fifteen to sixteen tissue types were collected from ten healthy adults(five males and five females).Total RNA was extracted,reverse-transcribed,and analyzed by qPCR.Gene stability was assessed using four algorithms(geNorm,NormFinder,BestKeeper,andΔCt)integrated with RefFinder,and the optimal gene number was determined using geNorm pairwise variation(V_(n/n+1)<0.15).Four transcriptome-derived genes(creld2,cd109,acy1,and miox)were used for validation.The C_(t)values of the five genes ranged from 15.47 to 20.83.β-actin and gapdh showed pronounced variability in expression stability among tissues and sexes,indicating their limited suitability for normalization.18S exhibited the highest expression(mean C_(t):15.47-16.29)and lowest variability but displayed sex-biased expression,whereas ef-1αand ef-1γremained consistently stable across most tissues in both sexes,with ef-1αbeing the most robust and showing no sex-related bias.Although specific rankings varied among tissues and sexes,the comprehensive results indicated that ef-1αand ef-1γpossessed the highest overall stability,followed by 18S,whileβ-actin and gapdh were the least stable.The final comprehensive rankings were ef-1γ>ef-1α>18S>gapdh>β-actin(male)and ef-1α>ef-1γ>18S>gapdh>β-actin(female).geNorm analysis(V2/3<0.15)indicated that two genes,mainly ef-1αand ef-1γ,were generally sufficient for reliable normalization in most tissues.Validation confirmed that normalization using the stable ef-1αand ef-1γaccurately reflected the expression differences among tissues,whereasβ-actin and gapdh can bias or confound statistical analyses.ef-1αand ef-1γare identified as the most reliable reference gene combination for qPCR analysis in S.japonica,while 18S can serve as an auxiliary gene for within-sex comparisons.The use ofβ-actin or gapdh alone is not recommended.This study establishes a systematic framework for selecting reliable reference genes in S.japonica,thereby facilitating robust qPCR normalization and providing a foundation for future gene expression research in S.japonica and other cephalopods.
文摘脂多糖(LPS)诱导的免疫应激下,选择适于校正肉鸡肝脏、心脏和回肠组织中目的基因表达量的内参基因。将30只1日龄的肉鸡随机分为对照组与处理组,21日龄时,处理组注射1 mg/kg的LPS,对照组注射等量灭菌生理盐水,2 h后,每组各取7只肉鸡的肝脏、心脏和回肠组织。通过实时荧光定量PCR技术(RT-q PCR)对各组织中β-肌动蛋白(ACTB)、β2-微球蛋白(B2M)、18 S核糖体RNA(18 S r RNA)、28 S核糖体RNA(28 S r RNA)和3-磷酸甘油醛脱氢酶(GAPDH)共5个内参基因的表达定量,利用Ref Finder和Ge Norm软件分析内参基因的稳定性及配对差异。结果显示:LPS刺激下,5个内参基因的表达水平有不同程度的变化。18 S r RNA、ACTB和GAPDH适于肝脏中目的基因表达量的校正;B2M和GAPDH适于心脏中目的基因表达量的校正;18 S r RNA和ACTB适于回肠中目的基因表达量的校正。选择表达稳定性高的内参基因适于校正目的基因的表达量,RT-q PCR应根据不同的研究对象进行选择,建议选择2个或2个以上的内参基因。
基金Supported by São Paulo Research Foundation(FAPESP),No.2010/08918-9 and 2020/11564-6the KBSP Young Investigator Fellowship,No.2011/00204-0+2 种基金the DBF Fellowship,No.2019/27492-7the LMG Fellowship,No.2014/01395-1the CFB Fellowship,No.2014/14278-3.
文摘BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.
文摘Quantitative real-time PCR(qRT-PCR)is widely used for investigating gene expression patterns and has many advantages,including its high sensitivity,fidelity,and specificity.Selecting a satisfactory internal reference gene is crucial for obtaining precise gene expression results in qRT-PCR analyses.In this study,the transcriptomic data of 2 potato varieties were screened for housekeeping genes with stable expression patterns.A total of 77 putative genes were selected,which were highly and stably expressed.Then,qRT-PCR analyses were performed to examine the expression levels of these 77 candidate reference genes in various potato tissues,including leaves,flowers,stolons,and tubers.Gene expression was represented by analyzing the Ct values at given threshold.Through geNorm and NormFinder program analyses,10 candidate genes with the most stable expression patterns were obtained,including RPL19,RPS15,RPS9,EF1α,TrxP1,RPS8,NTF,CAM,AACM,and RPS28.Moreover,through the comprehensive analyses of 4 statistical algorithms(i.e.,geNorm,NormFinder,BestKeeper,and RefFinder),results indicated that the most appropriate internal reference genes were RPL19 and EF1α.The obtained stable reference genes will contribute to future qRT-PCR analyses on potato tissue-related gene expression.
