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.     

The Use of Laser Microdissection in the Identification of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Human FFPE Epithelial Ovarian Tissue Samples

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.     

Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression

Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-α, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions.     

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。     

热胁迫下棉花粉蚧内参基因的筛选

【目的】筛选出热胁迫下棉花粉蚧Phenacoccus solenopsis的实时定量PCR最适内参基因。【方法】 本研究应用实时荧光定量PCR技术测定了棉花粉蚧2龄若虫、3龄若虫和雌成虫α-tub, β-tub, rpl32, GAPDH, SDHA和TBP共6个候选内参基因在7个不同温度处理（恒温18℃和 32℃, 以及37℃, 39℃, 41℃, 43℃和45℃热激处理1 h并在26℃恢复1 h）下mRNA表达水平的稳定性;应用geNorm, Bestkeeper, Normfinder和RefFinder软件分析6个基因表达的稳定性。【结果】 在不同高温胁迫下2龄若虫6个候选内参基因的稳定值M由小到大依次为α-tub（0.579）< GAPDH（0.654)< TBP（0.663）<β-tub（0.668）< rpl32（0.675）相似文献   

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。     

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase^plus software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results     

Validation of Reliable Reference Genes for Real-Time PCR in Human Umbilical Vein Endothelial Cells on Substrates with Different Stiffness

Background

The mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness.

Methodology/Principal Findings

Four algorithms, GeNorm^PLUS, NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes.

Conclusion/Significance:

Consequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study.     

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.     

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.     

Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction

Quantitative real-time RT-PCR (RT-qPCR) has proven to be a valuable molecular technique in gene expression quantification. Target gene expression levels are usually normalized to a stably expressed reference gene simultaneously determined in the same sample. It is critical to select optimal reference genes to interpret data generated by RT-qPCR. However, no suitable reference genes have been identified in human ovarian cancer to date. In this study, 10 housekeeping genes, ACTB, ALAS1, GAPDH, GUSB, HPRT1, PBGD, PPIA, PUM1, RPL29, and TBP as well as 18S rRNA that were already used in various studies were analyzed to determine their applicability. Totally 20 serous ovarian cancer specimens and 20 normal ovarian epithelial tissue specimens were examined. All candidate reference genes showed significant differences in expression between malignant and nonmalignant groups except GUSB, PPIA, and TBP. The expression stability and suitability of the 11 genes were validated employing geNorm and NormFinder. GUSB, PPIA, and TBP were demonstrated as the most stable reference genes and thus could be used as reference genes for normalization in gene profiling studies of serous ovarian cancer, while the combination of two genes (GUSB and PPIA) or the all three genes should be recommended as a much more reliable normalization strategy.