Validation of internal control for gene expression study in soybean by quantitative real-time PCR

Background  

Normalizing to housekeeping gene (HKG) can make results from quantitative real-time PCR (qRT-PCR) more reliable. Recent studies have shown that no single HKG is universal for all experiments. Thus, a suitable HKG should be selected before its use. Only a few studies on HKGs have been done in plants, and none in soybean, an economically important crop. Therefore, the present study was conducted to identify suitable HKG(s) for normalization of gene expression in soybean.     

Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR

For accurate and reliable gene expression results, normalization of real-time PCR data is required against a control gene, which displays highly uniform expression in living organisms during various phases of development and under different environmental conditions. We assessed the gene expression of 10 frequently used housekeeping genes, including 18S rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-1alpha, eIF-4a, and beta-TUB, in a diverse set of 25 rice samples. Their expression varied considerably in different tissue samples analyzed. The expression of UBQ5 and eEF-1alpha was most stable across all the tissue samples examined. However, 18S and 25S rRNA exhibited most stable expression in plants grown under various environmental conditions. Also, a set of two genes was found to be better as control for normalization of the data. The expression of these genes (with more uniform expression) can be used for normalization of real-time PCR results for gene expression studies in a wide variety of samples in rice.     

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.     

Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR

Quantitative real-time polymerase chain reaction (qRT-PCR) has been extensively used in several plant species as an accurate technique for gene expression analysis. However, the expression level of a target gene may be misconstrued due to unstable expression of the reference genes under different experimental conditions. Therefore, it is necessary to systematically evaluate these reference genes before experiments are conducted. Recently, more and more studies have focused on gene expression in pepper (Capsicum annuum L.). In this study, ten putative reference genes were chosen to identify expression stability by using geNorm and NormFinder statistical algorithms in ten different pepper sample pools, including those from different plant tissues (root, stem, leaf and flower) and from plants treated with hormones (salicylic acid and gibberellic acid) and abiotic stresses (cold, heat, salt and drought). EF1?? and UEP exhibited the most stable expression across all of the tested pepper samples. For abiotic stress or different hormone treatment, the ranking of candidate reference genes was not completely consistent, except for EF1?? which showed a relatively stable expression level. For different tissues, the expression of Actin1 was stable and it was considered an appropriate reference gene. It is concluded that EF1??, UEP and Actin1 are suitable reference genes for reliable qRT-PCR data normalization for the tissues and experimental conditions used in this experiment.     

Reference gene selection for normalizing gene expression using quantitative real-time PCR in Haemaphysalis longicornis

The Asian longhorned tick, Haemaphysalis longicornis, the dominant species of Ixodidae in Korea, has a wide distribution in East Asia, far-East Russia, and Western Pacific countries, and has recently been discovered in the Eastern states of the United States of America. H. longicornis transmits various pathogens, including Babesia ovate, Rickettsia japonica, and severe fever with thrombocytopenia syndrome virus (SFTSV). Considering its medical importance, in order to understand the physiology of H. longicornis, it is crucial to determine the expression of the genes of interest. Although quantitative real-time PCR (qRT-PCR) has been widely used to analyze gene expression, stably-expressed internal reference genes across samples of different conditions should be selected for the accurate normalization of target gene expression levels. Therefore, in this study, we investigated the expression levels of five candidate reference genes, namely ACT, RPP0, RPL23, TUB, and GAPDH, in H. longicornis under different conditions, including different collection months, developmental stages, and SFTSV infection status. Using four software programs, namely, NormFinder, BestKeeper, geNorm, and RefFinder, their expression stabilities were evaluated. Subsequently, a single gene between RPL23 and RPP0 was validated, which was found to be most stable reference gene after comparing the expression levels of HSP70 determined using different normalization methods.     

Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR

Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.     

荧光定量PCR检测干旱胁迫下长春花Crlea基因的相对表达

首次从长春花中克隆了Crlea（Crlea for Catharanthus roseus late embryogenesis abundant）的全长基因,采用荧光定量PCR方法对干旱胁迫下长春花叶片和根部Crlea基因的表达模式进行监测,结果表明,在0.5～8 h的胁迫时间中,叶片和根部的Crlea基因表现出相似的积累模式。长春花Crlea基因的表达随着胁迫时间的延长而表达增强。在叶片中,在6 h和8 h的干旱处理后,Crlea基因表达显著提高,分别是未处理材料的9.984和20.431倍。在根部, 在8 h的处理后,Crlea基因的表达量显著提高（2.831倍于对照)。初步结果表明Crlea基因的表达没有组织特异性,并且为干旱胁迫正调控。     

从ICR小鼠脂肪组织对resistin基因的扩增

目的 PCR扩增ICR小鼠脂肪组织的resistin基因,为进一步在ICR小鼠中开展resistin的相关研究提供必要的基础.方法 在脂肪组织中抽提总RNA,用RT-PCR进行resistin基因的体外扩增,经电泳检测和测序后用DNAMAN软件进行碱基序列分析.结果 抽提的总RNA完整性较好,RT-PCR产物电泳检测获...     

Short-term fatty acid intervention elicits differential gene expression responses in adipose tissue from lean and overweight men

The goal of this study was to investigate the effect of a short-term nutritional intervention on gene expression in adipose tissue from lean and overweight subjects. Gene expression profiles were measured after consumption of an intervention spread (increased levels of polyunsaturated fatty acids, conjugated linoleic acid and medium chain triglycerides) and a control spread (40 g of fat daily) for 9 days. Adipose tissue gene expression profiles of lean and overweight subjects were distinctly different, mainly with respect to defense response and metabolism. The intervention resulted in lower expression of genes related to energy metabolism in lean subjects, whereas expression of inflammatory genes was down-regulated and expression of lipid metabolism genes was up-regulated in the majority of overweight subjects. Individual responses in overweight subjects were variable and these correlated better to waist–hip ratio and fat percentage than BMI.