GDF-15 promotes mitochondrial function and proliferation in neuronal HT22 cells

The neuronal cell line HT22 is an excellent model for studying Parkinson's disease. Growth differentiation factor 15 (GDF15) plays a critical role in Parkinson's disease, but the molecular mechanism involved are not well understood. We constructed the GDF15 overexpression HT22 cells and detected the effects of overexpression of GDF15 on the viability, oxygen consumption, mitochondrial membrane potential of oligomycin-treated HT22 cells. In addition, we used a high-throughput RNA-sequencing to study the lncRNA and mRNA expression profiling and obtained key lncRNAs, mRNA, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathway. The expression of selected DElncRNAs was validated by quantitative real-time PCR (qRT-PCR). Our results showed that overexpression of GDF15 significantly reversed the cells viability, oxygen consumption, and mitochondrial membrane potential effect caused by oligomycin in HT22 cells. The 1093 DEmRNAs and 395 DElncRNAs in HT22 cells between GDF15-oligomycin non-intervention group and a normal control-oligomycin un-intervention group were obtained, and 394 DEmRNAs and 271 DElncRNAs in HT22 cells between GDF15-oligomycin intervention group and normal control-oligomycin intervention group were identified. Base on the GO and KEGG enrichment analysis of between GDF15-oligomycin intervention group and normal control-oligomycin intervention group, positive regulation of cell proliferation was most significantly enriched GO terms, and Cav1 was enriched in positive regulation of cell proliferation pathway. PI3K-Akt signaling pathway was one significantly enriched pathway in GDF15-oligomycin intervention group. The qRT-PCR results were consistent with RNA-sequencing, generally. GDF15 might promote mitochondrial function and proliferation of HT22 cells by regulating PI3K/Akt signaling pathway. Our study may be helpful in understanding the potential molecular mechanism of GDF15 in Parkinson's disease.     

植物实时荧光定量PCR内参基因的特点及选择

实时荧光定量PCR(qRT-PCR)具有灵敏度高、特异性强、重复的动态定量范围和高通量等优点,是进行植物基因表达和转录分析最常用的技术手段之一.选择合适的内参基因是正确运用实时荧光定量PCR分析目标基因表达变化的前提.近年来,大量研究表明,内参基因的选择应取决于研究者的实验条件;随着实验条件的变化,内参基因的选择也随之变化.因此,实时荧光定量PCR结果分析的准确性在很大程度上依赖于所选择的内参基因是否适合.该文从内参基因的选择、常用内参基因的特点、新内参基因的挖掘、应用内参基因组合的优点和内参基因的稳定性评价等几方面进行综述,以期为研究者在实验中选择合适的内参基因提供参考和理论依据.     

Evaluation of taxa-specific real-time PCR, whole-cell FISH and morphotaxonomy analyses for the detection and quantification of the toxic microalgae Alexandrium minutum (Dinophyceae), Global Clade ribotype

The dinoflagellate genus Alexandrium contains neurotoxin-producing species that have adversely affected the aquaculture industry in many countries. The morphological similarity between Alexandrium species has led to the development of molecular methods for the discrimination, enumeration and monitoring of toxic and nontoxic species. A quantitative real-time PCR assay (qRT-PCR) targeting the internal transcribed spacer 1-5.8S rRNA gene using hybridization probe technology was developed for the potentially toxic species Alexandrium minutum (Global Clade) (GC). The assay was specific with a detection limit of less than one cell equivalent. The assay was used to detect and quantify A. minutum (GC) in seawater samples collected during summer 2007 in Cork Harbour, Ireland. The results were compared with those obtained using whole-cell FISH (WC-FISH) and morphotaxonomy analyses. Alexandrium minutum did not reach high bloom concentrations over the sampling period (maximum of c . 6 × 10⁴ cells L⁻¹), and the average concentrations determined using qRT-PCR, WC-FISH and morphotaxonomy did not significantly differ in eight of nine comparisons. Regression curves showed positive relationships between the methods; WC-FISH and qRT-PCR slightly under- and overestimated, respectively, the A. minutum concentrations compared with the morphotaxonomy method. The qRT-PCR assay for A. minutum (GC) offers high-throughput sample analysis and may prove suitable for implementation in microalgae monitoring programmes and assist in population dynamics studies of the species.     

Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum

Research into diatom biology has now entered the post-genomics era, following the recent completion of the Thalassiosira pseudonana and Phaeodactylum tricornutum whole genome sequences and the establishment of Expressed Sequence Tag (EST) databases. The thorough exploitation of these resources will require the development of molecular tools to analyze and modulate the function of diatom genes in vivo. Towards this objective, we report here the identification of several reference genes that can be used as internal standards for gene expression studies by quantitative real-time PCR (qRT-PCR) in P. tricornutum cells grown over a diel cycle. In addition, we describe a series of diatom expression vectors based on Invitrogen Gateway technology for high-throughput protein tagging and overexpression studies in P. tricornutum. We demonstrate the utility of the diatom Destination vectors for determining the subcellular localization of a protein of interest and for immunodetection. The availability of these new resources significantly enriches the molecular toolbox for P. tricornutum and provides the diatom research community with well defined high-throughput methods for the analysis of diatom genes and proteins in vivo.     

小桐子生殖组织基因表达谱

虽然表达序列标签(ESTs,mRNA片段序列)已广泛用于高效基因发现和补充基因组注释的工作,最近,与实时荧光定量反转录PCR(qRT-PCR)结合,它也开始应用于种系遗传学、转录谱及其蛋白组学方面.通过对油料木本植物小桐子(J.curcas)的生殖组织基因表达水平的分析,预期可能找到一些与油脂合成相关的基因.这些研究成...     

Molecular and biochemical markers for monitoring dynamic shifts of cellulolytic protozoa in Reticulitermes flavipes

Lower termites rely on cellulolytic protozoa to aid in the digestion of their wood-based diet. However, despite the major contribution of protozoa to the lower termite digestive system, few techniques have been developed to monitor shifts in protozoan populations. This study investigated whether quantitative real-time PCR (qRT-PCR) and/or cellulase enzyme assays can be used to monitor changes of cellulolytic protozoan populations in the lower termite, Reticulitermes flavipes (Kollar). Previously developed cellulase primer sets were used to test for changes in cellulase gene expression, while three different cellulase enzyme assays were used to assess changes in cellulase enzyme activity. The results from this study indicate that qRT-PCR is a reliable method to monitor shifts in cellulolytic protozoan populations. Specifically, qRT-PCR can serve as a useful monitoring technique during high-throughput screening of novel termite control agents such as cellulase inhibitors, and help to answer questions relating to whether or not such control agents impact cellulolytic protozoan populations.     

Applying proteomics to signaling networks

The information from genome sequencing provides a new framework for a systems-wide understanding of protein networks and cellular function. Whereas microarray technologies provide information about global gene expression within cells, complementary proteomic strategies monitor expression of proteins and their posttranslational modifications. Improved technologies that have emerged for comprehensive and high-throughput protein analysis yield novel insights into cell regulation.     

施氏假单胞菌A1501铁吸收调节蛋白Fur的表达特性与功能鉴定

铁是细胞生理代谢的重要金属元素,是多种氧化还原酶类、过氧化物酶类的重要组成部分。革兰氏阴性菌中,胞内铁转运、储存和利用主要由铁吸收调节蛋白Fur（ferric uptake regulator）调控。水稻根际联合固氮菌——施氏假单胞菌（Pseudomonas stutzeri）A1501基因组中含有一个fur同源基因,但其具体功能尚不清楚。比较了A1501中fur基因及其编码蛋白的序列特征,并通过实时荧光定量PCR（real-time quantitative,qRT-PCR）和Western blotting杂交方法测定了fur基因和Fur蛋白的表达特性,结果发现fur基因表达水平与环境中铁浓度呈负相关,在铁限制条件下高表达,铁过量条件下表达受抑制。构建了fur基因突变株和功能回补株,表型测定发现fur突变影响了A1501的碳源代谢谱,并导致A1501对过氧化氢胁迫敏感。胞内铁含量测定表明,fur突变株内的铁含量为野生型的1.3倍,进一步qRT-PCR测定表明fur突变影响了铁离子转运系统及过氧化物酶编码基因的表达。研究结果为解析根际微生物胞内铁平衡调节及根际环境适应机制奠定了理论基础。     

Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR

Gene expression analysis provides significant insight to understand regulatory mechanisms of biology, yet acquisition and reproduction of quality data, as well as data confirmation and verification remain challenging due to a lack of proper quality controls across different assay platforms. We present a set of six universal external RNA quality controls for microbial mRNA expression analysis that can be applied to both DNA oligo microarray and real-time qRT-PCR including using SYBR Green and TaqMan probe-based chemistry. This set of controls was applied for Saccharomyces cerevisiae and Pseudomonas fluorescens Pf-5 microarray assays and qRT-PCR for yeast gene expression analysis. Highly fitted linear relationships between detected signal intensity and mRNA input were described. Valid mRNA detection range, from 10 to 7000 pg and from 100 fg to 1000 pg were defined for microarray and qRT-PCR assay, respectively. Quantitative estimation of mRNA abundance was tested using randomly selected yeast ORF including function unknown genes using the same source of samples by the two assay platforms. Estimates of mRNA abundance by the two methods were similar and highly correlated in an overlapping detection range from 10 to 1000 pg. The universal external RNA controls provide a means to compare microbial gene expression data derived from different experiments and different platforms for verification and confirmation. Such quality controls ensure reliability and reproducibility of gene expression data, and provide unbiased normalization reference for validation, quantification, and estimate of variation of gene expression experiments. Application of these controls also improves efficiency and facilitates high throughput applications of gene expression analysis using the qRT-PCR assay.