鳜鱼基因表达转录分析中的内参选择比较

目前基因表达的转录分析多采用单一或多个看家基因作为内参来校正目的基因的表达量。该实验以鳜鱼6个不同组织和5个不同胚胎发育阶段为研究对象,应用实时荧光定量PCR技术,观察了GAPDH、β-actin和18S rRNA三个看家基因mRNA水平的表达情况。geNorm统计分析表明,胚胎发育阶段β-actin表达最为稳定;不同的组织样品间,GAPDH表达最为稳定;而18S rRNA 的表达在不同的发育阶段不稳定。当利用多基因作为内参时,使用两个最稳定表达的看家基因即可对目的基因的表达进行准确校正。该结果证实了基因表达转录分析中内参基因选择的必要性,同时为鳜鱼等鱼类基因表达分析时内参基因的选择提供有价值的参考     

松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选

【目的】本研究拟选择适合用于分析松墨天牛Monochamus alternatus化学感受组织中基因表达的内参基因。【方法】依据转录组测序结果进行内参基因鉴定,利用RT-q PCR技术分析内参基因在松墨天牛不同发育阶段和不同性别化学感受组织间的表达差异,并利用软件ge Norm,Norm Finder和Best Keeper比较其表达的稳定性。【结果】松墨天牛转录组中鉴定出9个候选内参基因(Actin,TUB,18S rRNA,RPS27A,RPS3,RPL10,AK,GAPDH和EF1A),其中后7个候选内参基因在松墨天牛中被首次鉴定,松墨天牛候选内参基因和其他昆虫相应基因的同源性很高。9个候选内参基因引物均具有良好的扩增效率,18S rRNA的表达水平最高,EF1A的表达水平最低;18S rRNA和Actin在不同样品间的表达水平差异最大,GAPDH和TUB表达水平在不同样品间差异最小。ge Norm和Norm Finder软件分析认为,GAPDH是最稳定的内参基因,TUB是较为稳定的内参基因,18S rRNA和Actin是最不稳定的内参基因;Best Keeper软件分析认为,GAPDH和TUB是合适的内参基因,18S rRNA和Actin是不适合的内参基因。最适合校正松墨天牛化学感受组织中基因表达数据的内参基因数量为2个,即GAPDH和TUB,并且这样的内参基因组合可以用于不同发育阶段和不同性别的不同化学感受组织。【结论】本研究结果为利用RT-q PCR技术准确分析松墨天牛和其他天牛基因包括化学感受组织基因相对表达量的内参基因选择提供了重要参考。     

山羊不同组织器官的内参基因筛选

本研究通过比较9个内参基因在山羊不同组织中的表达水平进而确定最适合研究山羊组织表达的内参基因。本试验以简州大耳羊为试验材料,利用实时荧光定量PCR技术分析9个内参基因(GAPDH,PPIA,18S rRNA,PPIB,UXT,RPLP0,ACTB,EIF3K和TBP)在心脏、肝脏、脾脏、肺脏、肾脏、大肠、瘤胃、背最长肌和皮下脂肪等组织中的表达差异情况,并利用geNorm、NormFinder和BestKeeper等程序分析了它们的表达稳定性。geNorm和NormFinder程序一致显示TBP表达最稳定,其次是UXT和RPLP0;BestKeeper分析显示18S rRNA表达最为稳定,其次为TBP和ACTB;3个程序一致认为GAPDH表达稳定性最差。综合3个程序分析得出TBP最适合作为山羊组织中的内参基因,其次为UXT和RPLP0,GAPDH表达稳定性最差,不适合作为山羊组织内参,这为后续研究其他目的基因在山羊组织器官中的表达模式提供数据保障。     

光裸星虫(Sipunculus nudus)不同发育时期卵细胞内参基因的筛选

内参基因的选择对功能基因表达量的归一化处理尤为重要。为了筛选出光裸星虫不同发育时期卵子的最适内参基因,利用qRT-PCR测定了甘油醛-3-磷酸脱氢酶(GAPDH)、肽基脯氨酰顺反异构酶A(PPIA)、60S核糖体蛋白L10(60S-L10)、铁蛋白(Ferritin)、β-肌动蛋白(β-actin)、泛素C(UBC)、真核生物翻译起始因子(eIF)、NADH脱氢酶(NDH)、28S核糖体RNA(28S)、TATA盒结合蛋白(TBP)、18S核糖体RNA(18S)和琥珀酸脱氢酶A亚基(SDHA)共12个候选内参基因的表达水平,并通过4个程序(geNorm,NormFinder,BestKeeper以及RefFinder)综合分析了各基因的表达稳定性。结果显示:(1)12个候选内参基因均能获得特异性扩增产物,但表达情况各异;(2)对候选内参基因进行综合打分,得到候选内参基因稳定性排名为18S>GAPDH>28S>β-actin>UBC>e IF>NDH|TBP>PPIA|Ferritin>60S-L10>SDHA。18S和GAPDH稳定性较好,可作为不同发育时期卵细胞基因表达研究的单内参基因,或最优组合内参基因。     

翘嘴鳜per1 mRNA表达量分析中采用的内参基因稳定性比较

为筛选生物钟核心基因per1表达定量中的相对稳定性最好的内参基因,本研究取翘嘴鳜成鱼心脏、肝脏、肾脏、脑、红肌、白肌、肠、眼和脾等九个组织为研究对象,选取GAPDH、18S rRNA、β-actin、rps29、RPL13a、B2M和EF1a为内参基因,采用实时荧光定量PCR(qRT-PCR)对per1基因mRNA表达水平进行检测分析。研究结果表明18S rRNA和GAPDH的平均稳定值M最低,相对表达量最稳定。以18S rRNA和GAPDH为内参基因时分析发现per1基因表达量在肝脏中最高。本研究为在鱼类per1 mRNA表达检测过程中选用稳定的内参基因提供了实验和理论参考。     

基因表达转录分析中内参基因的选择

目前基因表达的转录分析多采用单一看家基因作为内参来校正目标基因的表达量.实验中以人肝癌BEL-7402细胞为研究对象,应用实时荧光定量PCR技术,观察了新型三肽化合物酪丝缬肽作用后RPL13A、UBC、EIF4A、B2M、GAPDH和ACTB共6个看家基因mRNA水平的表达情况.经过geNorm程序统计学分析处理,结果表明,这6个看家基因的表达存在差异,确定了RPL13A、UBC2个看家基因用于校正目标基因的表达量.基因表达转录分析中内参基因选择的必要性在实验中得以证明,更重要的是为各种实验因素影响下(尤其是新物质作用下)内参基因的选择介绍和提供了一种行之有效的方法.     

大灰象甲实时定量PCR内参基因的筛选

【目的】筛选出适合分析大灰象甲Sympiezomias velatus成虫不同组织中基因表达水平的内参基因。【方法】利用转录组测序技术获得大灰象甲管家基因序列作为候选内参基因,采用实时荧光定量PCR(qRT-PCR)技术分析候选基因在大灰象甲雌雄成虫触角、头、胸、腹和足中的表达量;并利用geNorm, NormFinder和BestKeeper软件及在线工具RefFinder评价候选基因的表达稳定性。以大灰象甲气味结合蛋白1(odorant bindng protein 1, OBP1)基因为目标基因验证候选基因在大灰象甲成虫不同组织中的表达稳定性。【结果】基于大灰象甲转录组数据首次鉴定得到β-肌动蛋白基因(ACT)、3-磷酸甘油醛脱氢酶基因(GAPDH)、18S核糖体RNA基因(18S rRNA)、60S核糖体蛋白L12基因(RPL12)、60S核糖体蛋白L32基因(RPL32)、40S核糖体蛋白S20基因(RPS20)、延伸因子2基因(EF2)、α-微管蛋白基因(TUA)和β-微管蛋白基因(TUB)共9个管家基因序列。geNorm分析结果显示,RPL12和RPS20是最稳定表达的内参基因,而BestKeeper和NormFinder分析结果显示最稳定表达的内参基因分别是TUA和TUB。综合各分析方法得出9个候选基因中TUB,TUA,RPS20和RPL12是最稳定表达的内参基因,而18S rRNA,ACT和GAPDH这3个广泛应用的内参基因则表现出最低的表达稳定性。最后以OBP1为目标基因对稳定性不同的4个候选基因进行稳定性验证,发现以TUB和RPL12为内参基因,OBP1在成虫不同组织之间的表达模式基本一致;而以RPL32为内参基因,表达模式与应用TUB作为内参基因时稍有不同,使用18S rRNA作为内参基因得到的OBP1表达模式则与应用TUB作为内参基因时的完全不一致。【结论】TUB,TUA,RPS20和RPL12可以作为分析大灰象甲成虫不同组织中基因表达水平的内参基因,为后续基因表达研究奠定了基础。     

牛转FAD3B基因胎儿成纤维细胞中内参基因的稳定性评估

采用RT-PCR扩增亚麻种子(Linum usitatissimum Linn)FAD3B基因,构建不同启动子的两种重组真核表达载体pIRES-AcGFP-CMV-FAD3B和pIRES-AcGFP-CAG-FAD3B,通过qRT-PCR检测转基因细胞中FAD3B基因的表达水平.以不同过表达水平的转基因细胞为试验对象,评价9种候选内参基因ACTB、GAPDH、18S rRNA、UXT、PPP1R11、RPS15A、SF3A1、EEF1A2和HMBS的稳定性.根据GeNorm、NornFinder和BestKeeper 3种统计学算法得到的稳定性值对基因进行排序.结果显示,内参基因稳定性的综合排序为PPP1R11＞EEF1A2＞1 8S rRNA＞RPS15A＞GAPDH＞HMBS＞UXT＞ACTB＞SF3A1,其中PPP1R11和EEF1A2是最稳定的内参基因.稳定内参的选择可以更加准确地校正基因的表达水平,从而为阐述基因的功能奠定了坚实的基础.     

朱红毛斑蛾实时荧光定量PCR内参基因的筛选

筛选朱红毛斑蛾Phauda flammans(Walker)在不同成虫组织、性别及发育阶段处理条件下稳定表达的内参基因,为进一步开展朱红毛斑蛾相关基因的定量研究提供参考.本研究以不同成虫组织(头、胸、腹、足、翅和触角)、不同成虫性别和不同发育阶段(卵、幼虫、蛹和成虫)为实验材料,对10个候选内参基因进行实时荧光定量PCR(qRT-PCR),并使用GeNorm、NormFinder和BestKeeper软件及RefFinder网站对候选内参基因的表达稳定性进行评价和综合分析.结果表明:在朱红毛斑蛾不同成虫组织基因定量研究中,TUB2＞GAPDH＞TUBJ＞AK＞EFlα＞ACTIN3＞TBP＞TUB3＞ACTIN2＞RPL32,建议以TUB2和GAPDH作为内参基因;在不同成虫性别基因定量研究中,TUB1＞EFlα＞ACTIN3＞RPL32＞ACTIN2＞TUB2＞AK＞GAPDH＞TUB3＞TBP,建议以 TUB1 和EFlα作为内参基因;在不同发育阶段基因定量研究中,ACTIN3＞TBP＞TUB1＞EFlα＞TUB3＞ACTIN2＞GAPDH＞RPL32＞TUB2＞AK,建议以ACTIN3和TBP作为内参基因.基于GeNorm分析,最佳内参基因使用数目为2个.     

微孢子感染东亚飞蝗实时定量PCR内参基因的筛选

【目的】筛选出微孢子Paranosema locustae感染条件下东亚飞蝗Locusta migratoria实时定量PCR最适内参基因。【方法】本研究应用实时荧光定量PCR技术测定东亚飞蝗甘油醛-3-磷酸脱氢酶基因(Glyceralde-hyde-3-phosphate dehydrogenase,GAPDH)、18s核糖体RNA(18s RNA)、微管蛋白基因(Tubulin,TUB)、肌动蛋白基因(Actin,ACT)和延伸因子1基因(Elongation factor1,EF1)5个候选基因在3个微孢子浓度(1×10~4、1×10~6和1×10~8个孢子/头)感染12 d后的表达稳定性;应用ge Norm、Bestkeeper和Normfinder 3个软件综合分析5个内参基因的稳定性。【结果】ge Norm软件对5个内参基因在不同微孢子虫浓度感染下稳定性分析结果表明:18S基因稳定性最低,平均表达稳定性值M值最高为0.658 4,TUB基因稳定性最高,M值最低为0.278 4。Norm Finder软件分析在不同微孢子虫浓度感染下的5个内参基因的稳定值分别为0.152(EF1)、0.181(ACT)、0.212(TUB)、0.329(GAPDH)和0.395(18S),可知内参基因稳定性顺序为:EF1>ACT>TUB>GAPDH>18S。由Best Keeper软件分析表明5个候选内参基因的SD值均小于1.0,表达均较稳定。根据其变异系数值CV的大小可知,ACT基因最为稳定。【结论】综合3种软件分析结果,在不同微孢子虫浓度感染下,可选择的较为稳定的内参基因ACT、EF1和TUB基因作为相对定量的参照基因。     

Effects of dimethyl sulfoxide and dexamethasone on mRNA expression of housekeeping genes in cultures of C2C12 myotubes

We used quantitative real-time RT-PCR to investigate the effects of dimethyl sulfoxide (DMSO) and dexamethasone (Dex) on the mRNA expression levels of the housekeeping genes β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), phosphoglycerate kinase 1 (PGK1), peptidylprolyl isomerase A (PPIA), and transferrin receptor (TFRC) in cultures of C2C12 myotubes. The ratios of ACTB mRNA levels to the HPRT1 mRNA level in C2C12 cells that were differentiating from myoblast cells to myotubes decreased from 0 to 120 h of culture, whereas the ratios of TFRC mRNA levels to the HPRT1 mRNA level increased from 0 to 120 h of culture. The ratios of GAPDH, GUSB, PGK1, and PPIA mRNA levels to the HPRT1 mRNA level remained constant from 0 to 120 h of culture. All housekeeping gene mRNA levels were unaffected by exposure to DMSO concentrations of 0.1% or less. The GAPDH mRNA level was increased by Dex, while the ACTB and PGK1 mRNA levels were significantly decreased by Dex. The GUSB, PPIA, and TFRC mRNA levels were unaffected by exposure to Dex. GUSB, HPRT1, and PPIA are thus suitable internal controls for evaluating mRNA expression levels in cultures of C2C12 cells.     

Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes

Quantitative real-time RT-PCR was used to investigate the effects of prototypical drug-metabolizing enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolylisomerase A (PPIA), TATA box binding protein (TBP), and transferrin receptor (TFRC) in primary cultures of cryopreserved human and rat hepatocytes. The mRNA levels of ACTB, GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in human hepatocytes were constant at all concentrations of inducers. However, the mRNA level of GAPDH relative to HPRT1 in rat hepatocytes was markedly increased by Rif. The mRNA levels of GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in rat hepatocytes were significantly increased by Dex. ACTB and HPRT1 are suitable internal controls for evaluating mRNA expression levels in primary cultures of human and rat hepatocytes after Rif, Dex, or Ome exposure.     

Evaluating reference genes to normalize gene expression in human epileptogenic brain tissues

Several reference genes have been used to quantify gene expression in human epilepsy surgery tissue. However, their reliability has not been validated in detail, although this is crucial in interpreting epilepsy-related changes of gene expression. We evaluated 12 potential reference genes in neocortical tissues resected from patients with temporal lobe epilepsy (TLE) with either few or many seizures (n=6 each) and post mortem controls (n=6) using geNorm and NormFinder algorithms. For all candidate reference genes threshold cycle (C(T)) values were measured. geNorm analysis revealed that the expression of e.g. glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl-transferase (HPRT) is unstable, whereas synaptophysin (SYP) and neuron-specific enolase (NSE)/mitochondrial 39S ribosomal protein L28 (MRPL) are most stably expressed. The geometric mean of SYP, NSE and MRPL levels is recommended as normalization factor (NF). NormFinder analysis, in contrast, indicated HPRT as the most stable single gene and recommended the geometric mean of TATA-box binding protein (TBP) and NSE levels as NF. Different values of upregulation of glial fibrillary protein (GFAP) expression were found in TLE tissue compared to control tissue depending on the NF used: 4.5-fold (geNorm-NF), 4.7-fold (NormFinder-NF), 4.2-fold (vs. GAPDH) and 7.8-fold (vs. HPRT). The expression of GABA(A) receptor subunit α5 (GARα5) was unaltered in the TLE groups compared to controls (geNorm-NF, NormFinder-NF, vs. GAPDH). However, normalization to HPRT suggests an apparent increase of GARα5 expression. In conclusion, the geNorm-NF (SYP/NSE/MRPL) and the NormFinder-NF (TBP/NSE) are equally suitable for normalization of gene expression in the human epileptogenic neocortex. In contrast, normalization to single and probably less stably expressed genes may not deliver accurate results.     

Identifying suitable reference genes for developing and injured mouse CNS tissues

Accurate quantification of gene expression is fundamental for understanding the molecular, genetic and functional bases of tissue development and diseases. Quantitative real‐time PCR (qPCR) is now the most widely used method of quantifying gene expression due to its simplicity, specificity, sensitivity, and wide quantification range. The use of appropriate reference genes to ensure accurate normalization is crucial for the correct quantification of gene expression from the early development, maturation, aging to injury processes in the central nervous system (CNS). In this study, we have determined the expression profiles of 12 candidate housekeeping genes (ACTB, CYC1, HMBS, GAPDH, HPRT1, RPL13A, YWHAZ, PPIA, RPLP0, TFRC, GUS, and 18S rRNA) in developing mouse brain and spinal cord. Throughout development, there was a significant degree of fluctuations in their expression levels, indicating the importance and complexity of finding appropriate reference genes. Three software including BestKeeper, geNorm and NormFinder were used to evaluate the stability of potential reference genes. GUS was the most stable gene and GUS/YWHAZ were the most stable reference gene pair across different developmental stages in different CNS regions, whereas HPRT1 and GAPDH were the most variable genes and thus inappropriate to use as reference genes. Therefore, our results identified GUS and YWHAZ as the best combination of two reference genes for expression data normalization in CNS developmental studies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 39–50, 2018     

Selection of Reference Genes for qRT-PCR in High Fat Diet-Induced Hepatic Steatosis Mice Model

With the epidemic proportions of obesity worldwide and the concurrent prevalence of hepatic steatosis, there is an urgent need for better understanding the intrinsic mechanism of hepatic steatosis, especially the changes of gene expression underlying the development of hepatic steatosis and its associated abnormal liver function. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible technique of gene expression analysis. However, for accurate and reliable gene expression results, it is vital to have an internal control gene expressed at constant levels under all the experimental conditions being analyzed for. In this study, the authors validated candidate reference genes suitable for qRT-PCR profiling experiments using livers from control mice and high fat diet-induced obese mice. Cross-validation of expression stability of ten selected reference genes using three popular algorithms, GeNorm, NormFinder, and BestKeeper found HPRT1 and GAPDH as most stable reference genes. Thus, HPRT1 and GAPDH are recommended as stable reference genes most suitable for gene expression studies in the development of hepatic steatosis.     

Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine

Gene expression studies in intestinal epithelial and stromal cells are a common tool for investigating the mechanisms by which the homeostasis of the small intestine is regulated under normal and pathological conditions. Quantitative real-time PCR (qPCR) is a sensitive and highly reproducible method of gene expression analysis, with expression levels quantified by normalization against reference genes in most cases. However, the lack of suitable reference genes for epithelial cells with different differentiation states and nonepithelial tissue cells has limited the application of qPCR in gene expression studies of small intestinal samples. In this study, 13 housekeeping genes, ACTB, B2M, GAPDH, GUSB, HPRT1, HMBS, HSP90AB1, RPL13A, RPS29, RPLP0,PPIA, TBP, and TUBA1, were analyzed to determine their applicability for isolated crypt cells, villus cells, deepithelialized mucosa, and whole mucosa of the mouse small intestine. Using geNorm and NormFinder software, GUSB and TBP were identified as the most stably expressed genes, whereas the expressions of the commonly used reference genes GAPDH, B2M, and ACTB, and ribosomal protein genes RPL13A, RPS29, and RPLP0 were relatively unstable. Thus, this study demonstrates that GUSB and TBP are the optimal reference genes for the normalization of gene expression in the mouse small intestine.     

Identification of Optimal Reference Genes for Gene Expression Normalization in a Wide Cohort of Endometrioid Endometrial Carcinoma Tissues

Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.     

Validation of internal controls for gene expression analysis in the intestine of rats infected with Hymenolepis diminuta

The non-invasive parasitic cestode Hymenolepis diminuta induces hypertrophy, hyperplasia and other changes in cell activity in the intestine of rats which are indicated in the expression of mRNA. We have investigated various house-keeping genes (GAPDH, β-actin, 18S and HPRT) and other internal controls (total RNA/unit biomass, total RNA/unit length of intestine) to validate gene expression in the rat intestine after cestode infection and drug-induced neuromodulation. Variation in GAPDH, β-actin, 18S and HPRT expression was observed in rat jejunal tissue according to treatment. Total RNA/unit length of intestine was found to be the most suitable internal control for normalizing target gene mRNA expression in both infected and/or drug-induced rat intestine. This normalization method may be applied to studies of gene expression levels in intestinal tissue where hypertrophy, hyperplasia, rapid growth and cell differentiation generally occur.