Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.     

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     

The normalization of gene expression data in melanoma: investigating the use of glyceraldehyde 3-phosphate dehydrogenase and 18S ribosomal RNA as internal reference genes for quantitative real-time PCR

We examined a panel of 26 melanoma and fibroblast samples (tissues and cultured cells) to evaluate the suitability of two commonly used housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA (rRNA), for quantitative real-time PCR. Both genes showed significant variations within the individual cell line and tissue groups. Although no overall trends were observed in the expression of the 18S rRNA, GAPDH was up-regulated in melanoma tissue and cultured cells compared with the corresponding normal samples. In melanoma and fibroblast cell lines and tissues, absolute quantification appears to be more appropriate than normalizing messenger RNA (mRNA) expression via GAPDH or 18S rRNA housekeeping genes.     

Identification and validation of reference genes for real time PCR expression studies in heads of nurse honeybees

The primary aim of this study was to identify reference genes and workers of particular role and ages that would be suitable for exploring genetic/epigenetic variations in constitutive expression of a gene encoding antimicrobial peptide defensin1 in worker heads using real-time PCR. This peptide is an integral component of larval food and honey and has potential to act against some brood pathogens. Expression levels of distinct genes may vary in worker heads due to genetic factors, age of bee, and particular role of a worker that depends on its age or colony needs. Prerequisite for exploring the variations in defensin1 expression was therefore to identify such workers in which correlated expression of defensin1 and suitable reference genes occurs. Selection process was done by carefully designed quantitative real-time PCR procedure in two colonies showing different age-related division of labor. Expression of ten candidate reference genes, defensin1 and amylase, as a marker of forager bees, was assessed in pooled head samples of workers aged 2 to 30 days. Correlated and moreover stable expression of defensin1 and six candidate genes was detected in nursing bees in both colonies. The suitable reference genes were therefore selected on the basis of their expression stability. This was evaluated by geNorm and NormFinder algorithms in pooled head samples and through plotted Cq data in head samples of individual nurse bees. As the best reference genes were selected: psa1, tctp1, cyclophilin, gapdh and mrjp4 (in this order). They are suitable for aforementioned defensin1 expression studies and also for studies of other genes expressed in heads of nurses. In addition, an amylase expression-based procedure for reliable distinguishing nurses from foragers was elaborated.     

Variation of Expression Levels of Seven Housekeeping Genes at Different Life-History Stages in Porphyra yezoensis

In order to identify the optimal internal control for relative real-time PCR when studying target gene expression in the red alga Porphyra yezoensis, we quantified the expression of seven housekeeping genes (18S ribosomal RNA, 30S ribosomal protein S8, Polyubiquitin-2, Glyceraldehyde-3-phosphate dehydrogenase, Elongation factor 1-alpha, Beta-tubulin and Actin 3) at different life-history stages. Absolute quantification was done by normalization to total RNA quantity and by normalization to genomic DNA quantity. We used these two normalization approaches, comparing the differences of expression levels of all candidate housekeeping genes between any two generations and across three life-history stages (filamentous sporophytes, leafy gametophytes and conchospores). We found GAPDH had the best stability in all cases and we recommend that GAPDH be considered as a potential internal control for gene expression studies at different life-history stages in P. yezoensis.     

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.     

Validation of reference genes for real-time quantitative PCR studies in gene expression levels of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> Zhang

Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.     

Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana.     

The Protective Effects of Achyranthes bidentata Polypeptides on Rat Sciatic Nerve Crush Injury Causes Modulation of Neurotrophic Factors

Pharmacological treatment is a therapeutic approach to improving nerve regeneration and functional recovery after peripheral nerve crush injury. The objective of the present study was to investigate the effects of the polypeptides isolated from Achyranthes bidentata Blume (abbreviated as ABPP) on rat sciatic crush injury and to test the possible involvement of neurotrophic factors. After surgical crush injury, rats received daily intraperitoneal injection of 0.2 ml saline containing 2 mg ABPP, 1 μg nerve growth factor (NGF) or no additive. The results from walking track analysis, electrophysiological assessment and histological evaluation indicated that the repair outcomes by ABPP treatment were close to those by NGF treatment, but better than those by treatment with saline alone. The quantitative real-time RT-PCR was used to monitor the mRNA expression of growth associated protein in the crush nerves and the mRNA expression of NGF, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine kinase (Trk)A and TrkB in the dorsal root ganglia (DRGs) at L4–L6. The mRNA expression of these genes in the crush nerve sample and DRGs sample was higher after treatment with ABPP or NGF than after treatment with saline alone. Our findings suggest that ABPP might protect peripheral nerve against crush injury through stimulating release of neurotrophic factors and the other cytokines.     

Selection of low-variance expressed Malus x domestica (apple) genes for use as quantitative PCR reference genes (housekeepers)

To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of reference genes relied on using homology to reference genes in model species. In this study, a genomics approach was used to identify apple genes with low variance in expression in 217 messenger RNA (mRNA)-seq data sets covering different tissues, during fruit development, and treated with a range of different stress conditions. Ten potential reference genes were chosen for validation by quantitative PCR (qPCR) over 29 different tissue types and treatments. From the combined mRNA-seq and qPCR results, three potential reference genes are proposed that can be used as good controls for PCR based expression studies. The three genes show homology to lipid transfer proteins, phytochrome protein phosphatase and the ubiquitination pathway. With the progression of research away from non-model species, this approach provides a robust method for selecting candidate genes for use as reference genes in qPCR.