Identification of Stably Expressed Reference Genes for RT‐qPCR Data Normalization in Defined Localizations of Cyclic Bovine Ovaries

Ovaries are highly complex organs displaying morphological, molecular and functional differences between their cortical zona parenchymatosa and medullary zona vasculosa, and also between the different cyclic luteal stages. Objective of the present study was to validate expression stability of twelve putative reference genes (RGs) in bovine ovaries, considering the intrinsic heterogeneity of bovine ovarian tissue with regard to different luteal stages and intra‐ovarian localizations. The focus was on identifying RGs, which are suitable to normalize RT‐qPCR results of ovaries collected from clinical healthy cattle, irrespective of localization and the hormonal stage. Expression profiles of twelve potential reference genes (GAPDH, ACTB, YWHAZ, HPRT1, SDHA, UBA52, POLR2C, RPS9, ACTG2, H3F3B, RPS18 and RPL19) were analysed. Evaluation of gene expression differences was performed using genorm , normfinder , and bestkeeper software. The most stably expressed genes according to genorm , normfinder and bestkeeper approaches contained the candidates H3F3B, RPS9, YWHAZ, RPS18, POLR2C and UBA52. Of this group, the genes YWHAZ, H3F3B and RPS9 could be recommended as best‐suited RGs for normalization purposes on healthy bovine ovaries irrespective of the luteal stage or intra‐ovarian localization.     

Screening of the Reference Genes for the Study of Peripheral Blood Mononuclear Cells by Quantitative Real-time PCR in Min Pigs

Min pig is a local pig breed in Northeast China. It has been well-adapted to the local cold weather,but few genes related to its environmental adaptation have been studied. For studies about environmental adaptation of Min pig on molecular level,it is important to have proper reference genes for quantification of gene expression by quantitative Real-time PCR. In this study,12 reference genes (B2M,ACTB,RPL11,RPL4,YWHAZ,GAPDH,HPRT1,SDHA,HMBS,IDH3B,TUBB2B and TBP1) were evaluated for their potential as the reference gene in Min pig peripheral blood mononuclear cells under different temperatures. Blood samples were collected from 3 Min pigs which were under -25,5,10 and 30℃,respectively. Mononuclear cells were separated using density gradient centrifugation. Statistical algorithms including geNorm,Normfinder and BestKeeper were employed to assess the stabilities of these genes. Analysis of geNorm and Normfinder revealed that all these 12 genes were highly stable. However,ACTB,GAPDH,SDHA,HPRT1,TBP1 and YWHAZ genes (SD<1) were found to be more stable than other six genes (SD>1),of which TBP1 was the most stable one using BestKeeper program. To summarize,ACTB,GAPDH,SDHA,HPRT1, TBP1 and YWHAZ genes were suitable to be the reference genes,with TBP1 was the best one.     

Reference gene selection for reverse transcription quantitative polymerase chain reaction in chicken hypothalamus under different feeding status

This study was designed to investigate the stability of 10 candidate reference genes, namely ACTB, B2M, GAPDH, HMBS, LBR, POLR2B, RN18S, RPS17, TBP, and YWHAZ for the normalization of gene expression data obtained by quantitative real‐time polymerase chain reaction (qPCR) in studies related to feed intake of chicken. Samples were isolated from hypothalamus under three different nutritional status (ad libitum, fasted for 24 hr, fasted for 24 hr then refed for 2 hr). Five different algorithms were applied for the analysis of reference gene stability: BestKeeper, geNorm, NormFinder, the comparative ΔCt method, and a novel approach using multivariate linear mixed‐effects modelling for stable reference gene selection. TBP and POLR2B were identified as the two most suitable and B2M and RN18S as the two least stable reference genes for normalization. Despite our review, the current literature showing that RN18S is one of the most commonly used reference gene in chicken gene expression studies, its applicability for normalization should be evaluated before each qPCR experiment.     

民猪外周血单核细胞实时荧光定量PCR内参基因的筛选

民猪是能够适应东北地区寒冷环境的地方猪种,但目前对其环境适应性相关基因的研究较少,也没有确定适合此研究所需的实时荧光定量PCR的内参基因。因此,本试验通过研究常用的12个内参基因（B2M、ACTB、RPL11、RPL4、YWHAZ、GAPDH、HPRT1、SDHA、HMBS、IDH3B、TUBB2B和TBP1）在不同环境温度下民猪外周血单核细胞中的表达稳定性,旨在确定合适的内参基因进行相关研究。分别在-25、5、10和30℃采集3头民猪耳静脉血分离出单核细胞,利用geNorm、NormFinder、BestKeeper 3种软件分析12个候选内参基因的Ct值,筛选出表达稳定的基因作为内参基因。经geNorm和NormFinder计算获得各候选基因的M值发现,12个候选基因的表达均相对稳定,而BestKeeper的分析则显示ACTB、GAPDH、SDHA、HPRT1、TBP1、YWHAZ基因的SD值均<1,可用作本研究条件下的内参基因,而另外6个基因SD值则>1,不符合作为内参基因的标准。综合3种分析的结果,在本研究条件下,TBP1基因的稳定性最高,ACTB、GAPDH、SDHA、HPRT1和YWHAZ基因也符合作为内参基因的标准,都可研究在不同环境温度下民猪外周血单核细胞中基因表达时作为内参基因。     

Reference gene screening for analyzing gene expression in the heart,liver, spleen,lung and kidney of forest musk deer

The screening of reference genes for real-time quantitative PCR (qPCR) in forest musk deer (FMD) tissue is of great significance to the basic research on FMD. However, there are few reports on the stability analysis of FMD reference genes so far. In this study, We used qPCR to detect the expression levels of 11 reference gene candidates (18S rRNA, beta-actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box-binding protein [TBP], hypoxanthine phosphoribosyltransferase 1 [HPRT1], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide [YWHAZ], hydroxymethylbilane synthase [HMBS], eukaryotic translation elongation factor 1 alpha 1 [EEF1A1], succinate dehydrogenase complex flavoprotein subunit A [SDHA], peptidylprolyl isomerase B [PPIB], and ubiquitin C [UBC]) in heart, liver, spleen, lung and kidney of FMD. After removing 18S rRNA on account of its high expression level, geNorm, NormFinder, BestKeeper and ΔCt algorithms were used to evaluate the expression stability of the remaining genes in the five organs, and further comprehensive ranking was calculated by RefFinder. According to the results, the selected reference genes with the most stable expression in the heart of FMD are SDHA and YWHAZ, while in the liver are ACTB and SDHA; in the spleen and lung are YWHAZ and HPRT1; in the kidney are YWHAZ and PPIB. The use of common reference genes in all five organs is not recommended. The analyses showed that tissue is an important variability factor in genes expression stability. Meanwhile, the result can be used as a reference for the selection of reference genes for qPCR in further study.     

Identification of Stable Reference Genes for Gene Expression Studies Using Quantitative Real Time PCR in Buffalo Oocytes and Embryos

The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15, RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes/embryos. The information would help in more accurate interpretation of gene expression data from oocytes/embryos towards understanding the molecular events in these cells during development.     

Reference genes for canine skin when using quantitative real-time PCR

Quantitative real-time PCR (qPCR) facilitates the quantification of mRNA expression. Accurate qPCR analysis of gene expression requires the normalisation of data using a reference or housekeeping gene which is expressed at a similar level in all tissues tested. GAPDH is the most well known and most widely used reference gene but many papers have demonstrated that it is not stably expressed in different tissues. The aim of this study was to measure reference gene stability in canine skin using real-time qPCR. Skin samples from healthy control dogs (n=7) and dogs with atopic dermatitis (lesional skin n=7 and non-lesional skin n=7) were used to quantify seven reference genes (IMP, CG14980, S7, HIRA, GAPDH, RPL13A and SDHA) in canine whole skin. Three different statistical programs (Bestkeeper, GeNorm and Normfinder) were used to assess the stability of the reference genes. The results confirmed that GAPDH is not a stably expressed reference gene in canine skin; this finding may influence interpretation of previous qPCR studies on canine skin using this as a reference gene. RPL13A and CG14980 were found to be the most stably expressed genes in canine whole skin and would be more suitable as reference genes in future studies.     

白羽王鸽不同组织RT-qPCR内参基因的筛选

内参基因的正确选择是获得精准、可靠RT-qPCR数据的重要前提。本研究随机选取3只28日龄的白羽王鸽,采集心、肝、脾、肺、肾、胸肌、腿肌、腹脂、卵巢9个组织作为实验材料,选取GAPDH、β-actin、18S rRNA和RPS24个常见的内参基因作为候选内参基因。利用RT-qPCR技术和geNorm、NormFinder、BestKeeper软件及Ct值分析法对候选基因在不同组织中的表达进行检测和稳定性分析。结果表明:geNorm、NormFinder软件分析与Ct值分析法的结果一致,即18S rRNA基因稳定性最佳,RPS2次之;BestKeeper软件的分析结果显示RPS2基因表达稳定性最好,18S rRNA基因次之。因此,推荐选择RPS2+18S rRNA的内参基因组合用于白羽王鸽不同组织基因表达的研究。     

Screening of Reference Genes for Gene Expression in Layer Hens Tissues Using Real-time Quantitative PCR

To screen the reference genes of stable expression in different tissues of Hy-line layer (Gallus domesticus), RPS2, β-actin, GAPDH and HMBS genes were chosen as candidate gene, the software of geNorm, NormFinder and the Ct value were employed to analyze their stability in eight tissues (heart, liver, lung, kidney, duodenum, tibia, pancreas and uterus) of 120 days of age layers using Real-time quantitative PCR technology. The results showed that RPS2 gene was always dominant stability by the analysis of Ct value and geNorm software, the β-actin gene was followed; The NormFinder software showed that the HMBS gene was relative stability, and HMBS and RPS2 genes were the optimal combination; When analyzed the expression of constant reference gene in different tissues, it found that the most stable reference gene in different tissues was not the same. Therefore, it concluded that different tissues had different optimal gene, but they had the common potential, RPS2 and β-actin genes had the relative expression stability comparing the other gene.     

实时荧光定量PCR研究蛋鸡组织基因表达的内参基因筛选

为筛选出蛋鸡不同组织中稳定表达的内参基因,试验以120日龄的海兰灰蛋鸡（Gallus domesticus）为试验动物,选取常见内参基因RPS2、β-actin、GAPDH和HMBS作为候选基因,利用实时荧光定量PCR技术Ct值分析法对4个候选内参基因的相对表达进行测定,利用独立评价软件geNorm和NormFinder对蛋鸡的4个候选内参基因在不同组织（心脏、肝脏、肺脏、肾脏、肠、胫骨、胰脏和子宫）中的表达稳定性进行评价。结果显示,Ct值分析和geNorm程序分析均得出相似的结果,即RPS2基因始终占主导地位,稳定性最强,β-actin基因次之;NormFinder软件分析显示,HMBS基因稳定性最好,最佳组合为HMBS和RPS2基因;当分析不同组织中表达恒定的内参基因时,发现不同组织最稳定的内参基因也不完全相同。由此说明,不同的组织器官和不同的分析方法得出的结论虽然不完全一致,但它们却有着一定的潜在共性,发展趋势有一定相似性,即RPS2和β-actin基因相对稳定性更强。     

Developmental competence of heat stressed oocytes from Holstein and Limousine cows matured in vitro

The negative effects of heat stress on dairy cattle's fertility have been extensively studied, but the relevant knowledge for beef cattle is rather limited. The aims of this study were to investigate the effects of HS during in vitro maturation on the developmental potential of oocytes derived from Limousine and Holstein cows and to estimate the effect of the differential gene expression of important genes in oocytes, cumulus cells and blastocysts in the growth competence between the breeds. In seven replicates, cumulus oocyte complexes from Holstein and Limousine cows were matured for 24 hr at 39°C (controls C; Hol_39, Lim_39) or at 41°C from hour 2 to hour 8 of IVM (treated T; Hol_41, Lim_41), fertilized, and presumptive zygotes were cultured for 9 days at 39°C. Cleavage and embryo formation rates were evaluated 48 hr post-insemination and on days 7, 8 and 9, respectively. From all groups, subsets of cumulus cells, oocytes and blastocysts were analysed for the relative expression of genes related to metabolism, stress, apoptosis and placentation. No difference was detected in cleavage rate or in blastocyst formation rate among the control groups. In both breeds, heat stress reduced blastocyst yield, but at all days the suppression was higher in Limousines. In Holsteins, altered gene expression was detected in cumulus cells (G6PD, GLUT1) and blastocysts (PLAC8), while in Limousines, differences were found in oocytes (G6PD, HSP90AA1), in cumulus cells (CPT1B, HSP90AA1, SOD2) and blastocysts (DNMT, HSP90AA1, SOD2). It appears that Holstein COCs are more tolerant than Limousine COCs, possibly due to compulsory, production driven selection.     

Development of gene expression assays measuring immune responses in the spotted hyena (Crocuta crocuta)

As scavengers, spotted hyenas (Crocuta crocuta) are exposed to a wide array of pathogens but exhibit low mortality rates due to infectious disease. This suggests that this species exhibits a unique and robust immune response to pathogens. However, few tools exist to measure cell-mediated immunity (CMI) in hyenas and we aimed to develop a gene expression assay to quantify antigen-specific responses. Whole blood from five Mycobacterium bovis- sensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genes ACTB, GAPDH, YWHAZ and TBP in these samples was determined as well as the mean fold change in the expression of IFNG, CXCL8, CXCL9, CXCL10 and CXCL11 in M. bovis-antigen stimulated blood. The expression of YWHAZ and TBP showed greatest stability, and YWHAZ was selected as a reference for further analysis. The expression of CXCL9 and CXCL11 showed greatest upregulation in antigen-stimulated blood and the assay results for these genes were strongly correlated. The measurement of antigen-induced CXCL9 and CXCL11 expression, relative to that of YWHAZ, can be used to measure CMI responses to infectious diseases in spotted hyenas.     

Pluripotency‐associated genes reposition during early embryonic developmental stages in pigs

We examined the allelic expression and positioning of two pluripotency‐associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4‐ and 8‐cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi‐allelically increased from 45% at the 4‐cell stage to 60% at the 8‐cell stage. Moreover, in 8‐cell embryos, SOX2 was expressed bi‐allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4‐ to 8‐cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4‐ or 8‐cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency‐associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.