Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or house- keeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rp113α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rp113α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

Reference genes identified in the silkworm Bombyx moil during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR

Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction （qRT-PCR）, require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT- PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 （represented by sw22934, sw14876, and sw13956） were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin （sw22671）, the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.     

Defining reference genes for quantitative real-time PCR analysis of anther development in rice

Quantitative real-time polymerase chain reaction （qPCR） is one of the most accurate and widely used methods for gene expression analysis. However, the choice of reference genes for normalization is critical for accurate quantifica- tion of gene expression. As development of genomics, mining large-scale datasets such as microarray and RNAsequencing data becomes a new approach for exploitation of new reference genes. In this study, we analyzed an RNAsequencing dataset of rice anther and 167 microarray datasets involving different tissues and developing stages of rice anthers and pollens. We selected 12 candidate genes and other 5 reference genes, including ACT1, eEF-1α, GAPDH, Exp2, and CCDC72 used in previous studies, and evaluated their expression in eight tissues and different developmental stages of anthers in rice variety 9311 and Yuetai. UPF3, elF4A-3, GAPDH, and PPP6 were identified as the most suitable reference genes for qPCR analysis of anther development in rice. The new candidate reference genes showed more stable expression than the traditionally used reference genes. These results provide a set of reliable reference genes for studies in rice anther developmental process.     

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

Although the accuracy of quantitative real-time polymerase chain reaction （qRT-PCR） is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing （RNA-Seq） to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression proffies of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes （B2M, GAPDH, GUSB, HMBS, HPRT1, IP08, PGK1, POLR2A, PPIA, RPLPO, TBP, TFRC, UBC, and YWHAZ）, the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes （ACTB） and the 5 additional candidate reference genes （EEFIA1, FAU, RPS9, RPSll, and RPSI4） were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEFIAI, FA U, RPS9, RPSl l, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.     

A hybrid SOM-SVM approach for the zebrafish gene expression analysis

Microarray technology can be employed to quantitatively measure the expression of thousands of genes in a single experiment. It has become one of the main tools for global gene expression analysis in molecular biology research in recent years. The large amount of expression data generated by this technology makes the study of certain complex biological problems possible, and machine learning methods are expected to play a crucial role in the analysis process. In this paper, we present our results from integrating the self-organizing map （SOM） and the support vector machine （SVM） for the analysis of the various functions of zebrafish genes based on their expression. The most distinctive characteristic of our zebrafish gene expression is that the number of samples of different classes is imbalanced. We discuss how SOM can be used as a data-filtering tool to improve the classification performance of the SVM on this data set.     

Improve survival prediction using principal components of gene expression data

The purpose of many microarray studies is to find the association between gene expression and sample characteristics such as treatment type or sample phenotype. There has been a surge of efforts developing different methods for delineating the association. Aside from the high dimensionality of microarray data, one well recognized challenge is the fact that genes could be complicatedly inter-related, thus making many statistical methods inappropriate to use directly on the expression data. Multivariate methods such as principal component analysis （PCA） and clustering are often used as a part of the effort to capture the gene correlation, and the derived components or clusters are used to describe the association between gene expression and sample phenotype. We propose a method for patient population dichotomization using maximally selected test statistics in combination with the PCA method, which shows favorable results. The proposed method is compared with a currently well-recognized method.     

Regulated genes in mesenchymal stem cells and gastric cancer

AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.     

Evaluation of the stability of standard reference genes of adipose-derived mesenchymal stem cells during in vitro proliferation and differentiation

Mesenchymal stem cells (MSCs) from a variety of sources are being used in pre-clinical and clinical studies. The choice of optimal source for treatment of diseases requires quantitative evaluation of self-renewal, proliferation and differentiation potencies of MSCs. For this purpose, quantitative real-time polymerase chain reaction (qRT-PCR) technique is used to determine the expression of genes. qRT-PCR requires the normalization of the gene expression levels by the use of reference genes in order to obtain accurate and reliable results. There is a limited number of studies focused on the selection of reference genes that are appropriate and reliable for MSCs. Thus, no single reference gene has yet been found for use in the in vitro proliferation and differentiation of MSCs. The aim of this study is to investigate the stability of the expression of widely used reference genes during the in vitro proliferation and differentiation of human adipose-derived mesenchymal stem cells (hASCs). For this purpose, 13 reference genes commonly used in MSC studies were selected. As a result, the expression stabilities of EF1α, RPLP0 and RPL13A genes were found to be high and were predicted to be suitable for use as reference genes for normalization in hASC studies. The GAPDH was identified as the gene with the lowest expression stability and evaluated to be an unsuitable reference gene for hASC differentiation studies. This piece of information could be crucial for the selection of appropriate reference genes and accurate measurement of gene expression in hASC studies.

     

Evaluation of suitable reference gene for real-time PCR in human umbilical cord mesenchymal stem cells with long-term in vitro expansion

In real-time quantitative PCR, the accuracy of normalized data is highly dependent on the stability of the reference genes. However, reference gene expression in a given cell type or experimental condition can vary considerably. The goal of this study was to establish a reliable set of reference genes for real-time PCR studies using human umbilical cord mesenchymal stem cells with long-term in vitro expansion. The stability of ten potential reference genes was examined in human umbilical cord mesenchymal stem cells. We found that Ywhaz and Rpl13a, not beta-actin or Gapdh, were the most stably expressed of the internal control genes in different passages of human umbilical cord mesenchymal stem cells. Ywhaz and Rpl13a could be used as reference genes for relative gene quantification and normalization purposes in real-time PCR studies of human umbilical cord mesenchymal stem cells.     

Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax)

The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), β-actin (two regions of β-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), β2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization.     

实时荧光定量PCR分析中毛果杨内参基因的筛选和验证

实时荧光定量PCR(qRT-PCR)技术具有高灵敏性、高保真性和高特异性, 被广泛应用于基因表达的分析。在数据处理过程中, 选用稳定表达的基因作为内参基因对准确分析实验结果非常关键。以毛果杨(Populus trichocarpa)的不同组织以及锌胁迫下的组培苗为材料, 使用荧光定量PCR方法分析了TUA8、TUB6、ubiquitin、GAPDH、actin、18S rRNA和EF1α 7个看家基因的表达情况。通过geNorm、NormFinder和BestKeeper 3个程序的综合分析, 发现actin、ubiquitin、EF1α和18S rRNA的稳定性较好, 可用作毛果杨基因表达研究的内参基因; 而TUB6在不同组织中稳定性最差; GAPDH在锌胁迫下的组织中稳定性最差, 因此不适宜作为内参基因。毛果杨NAC基因的表达分析, 进一步验证了上述结果。该研究对采用qRT-PCR方法分析毛果杨基因表达过程中内参基因的选择具有指导作用, 同时对揭示NAC基因的功能也有一定的意义。     

Bt毒素诱导下小菜蛾实时定量PCR 内参基因的筛选

【目的】筛选出Bt毒素诱导后的小菜蛾Plutella xylostella (L.)的实时定量PCR最适内参基因。【方法】选取核糖体18S rRNA (18S rRNA)、 肌动蛋白（ACTB）、 延伸因子（EF1）、3-磷酸甘油醛脱氢酶（GAPDH）、 核糖体蛋白L32 (RPL32)、 核糖体蛋白S13 （RPS13）、 核糖体蛋白S20 （RPS20）和β-微管蛋白(TUB)基因作为候选内参基因, 以geNorm、 Normfinder和BestKeeper软件分析这8个基因在Bt毒素诱导后的小菜蛾不同品系中肠组织中的表达稳定性。并应用筛选出来的内参基因分析小菜蛾氨肽酶2（aminopeptidase N2, APN2）基因的表达水平。【结果】geNorm软件以RPS13和EF1为最稳定内参基因, NormFinder和BestKeeper软件均以RPS13和RPL32为最稳定基因。使用3种不同内参基因分析Bt毒素诱导后的小菜蛾Bt抗性和敏感品系中ANP2表达水平时, 新的内参基因EF1和传统内参基因RPL32表现了良好的稳定性, 二者作为标准化因子, ANP2表达量结果基本一致, 而使用18S rRNA作为内参基因, 却导致部分表达量分析结果有所误差。【结论】筛选出PRS13,RPL32和EF1可以作为小菜蛾某些试验条件下的内参基因, 对小菜蛾基因表达研究奠定了一定基础, 也对其他昆虫内参基因的筛选具有参考价值。     

Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.