Expression of Transferrin mRNA in the CNS of Normal and Jimpy Mice

Both the iron mobilization protein transferrin and iron itself are found predominantly in oligodendrocytes in the brain and consequently have been hypothesized to have a role in myelination. This study is designed to begin to understand the mechanism(s) that control the expression of transferrin at the gene level in the nervous system using a hypomyelinating murine mutant (jimpy mouse). With this animal model it is possible to determine if transferrin gene expression in the nervous system is dependent on the presence of a mature oligodendrocytic population. The results demonstrate that normally expression of the transferrin gene increases from postnatal day 5 to 22-25 and then levels off in the adult. In the jimpy mouse, the relative amount of transferrin gene expression is less than that of littermate controls at 5 days of age. Furthermore, transferrin gene expression does not increase with age beyond the level observed at postnatal day 5 in the jimpy mouse. It is concluded from this study that the majority of the transferrin mRNA in the mouse brain is expressed by and/or requires the presence of a mature oligodendrocytic population.     

Quality Assessment and Data Analysis for microRNA Expression Arrays

MicroRNAs are small (~22 nt) RNAs that regulate gene expression and play important roles in both normal and disease physiology. The use of microarrays for global characterization of microRNA expression is becoming increasingly popular and has the potential to be a widely used and valuable research tool. However, microarray profiling of microRNA expression raises a number of data analytic challenges that must be addressed in order to obtain reliable results. We introduce here a universal reference microRNA reagent set as well as a series of nonhuman spiked-in synthetic microRNA controls, and demonstrate their use for quality control and between-array normalization of microRNA expression data. We also introduce diagnostic plots designed to assess and compare various normalization methods. We anticipate that the reagents and analytic approach presented here will be useful for improving the reliability of microRNA microarray experiments.     

Integrative Analysis of the Expression of microRNA,Long Noncoding RNA,and mRNA in Osteoarthritis and Construction of a Competing Endogenous Network

Biochemical Genetics - This study aimed to identify potential core microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and mRNAs in osteoarthritis (OA) to construct a competing endogenous RNA...     

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.     

野生型小鼠皮肤cyclinA1 mRNA的表达及其意义

目的从RNA水平探讨cyclinA1在野生型小鼠皮肤中的表达及意义。方法选取30只大于6个月的野生型小鼠,用原位杂交方法定位检测cyclinA1 mRNA在头颈部皮肤中的表达,同时以不加探针皮肤标本作为阴性对照,另取雄性小鼠睾丸组织作为阳性对照。结果分别有25只野生型小鼠在皮脂腺部位及12只在表皮全层有cyclinA1 mRNA表达。其中,皮脂腺部位强阳性及阳性表达分别占50.0%和33.3%,阳性率为83.3%;表皮全层强阳性及阳性表达分别占13.3%和26.7%,阳性率为40.0%。皮脂腺阳性率显著高于表皮(P0.05)。结论CyclinA1 mRNA在野生型小鼠头颈部皮肤的皮脂腺部位及表皮全层的表达均有较高的阳性率,尤其皮脂腺表达的阳性率更高。     

拟南芥热激转录因子耐高温功能分析

从拟南芥基因组中克隆了热激转录因子(At Hsf A6a),构建了过量表达(over-expression,OE)和反义(anti-sense,AS)植物表达载体并转化拟南芥,获得了拟南芥纯合转基因株系。对其进行耐高温处理,结果显示:43℃处理2 h,过量表达转基因植株存活率(86%)远高于野生型(59%);而反义转基因植株存活率则只有43%,显著低于野生型。43℃处理0.5 h,过量表达转基因植株的离子渗漏水平显著低于野生型,而反义转基因植株则大幅度升高。基因表达分析证明,AtHsfA6a的表达受热胁迫诱导,并且Hsp70是受AtHsfA6a调控的下游靶基因。上述结果表明,拟南芥AtHsfA6a可能通过调节Hsp70表达,提高植物耐受高温胁迫的能力。     

Integration of mRNA Expression Profile,Copy Number Alterations,and microRNA Expression Levels in Breast Cancer to Improve Grade Definition

Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number.     

Involvement of microRNA in AU-rich element-mediated mRNA instability

AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.     

Cell-free seminal mRNA and microRNA exist in different forms

Background

The great interest in cell-free mRNA, microRNA (miRNA) as molecular biomarkers for clinical applications, and as ‘signaling’ molecules for intercellular communication highlights the need to reveal their physical nature. Here this issue was explored in human cell-free seminal mRNA (cfs-mRNA) and miRNA (cfs-miRNA).

Methodology/Principal Findings

Selected male reproductive organ-specific mRNAs, miRNAs, and piRNAs were quantified by quantitative real-time PCR in all experiments. While the stability of cfs-miRNA assessed by time-course analysis (up to 24 h at room temperature) was similar with cfs-mRNA, the reductive changes between cfs-miRNA and cfs-mRNA after filtration and Triton X-100 treatment on seminal plasma were very different, implying their different physical nature. Seminal microvesicles (SMVs) were then recovered and proportions of cfs-mRNA and cfs-miRNA within SMVs were quantified. The amounts of SMVs- sequestered cfs-mRNAs almost were the same as total cfs-mRNA, and were highly variable depending on the different sizes of SMVs. But most of cfs-miRNA was independent of SMVs and existed in the supernatant. The possible form of cfs-miRNA in the supernatant was further explored by filtration and protease K digestion. It passed through the 0.10-µm pore, but was degraded dramatically after intense protease K digestion.

Conclusions/Significance

The predominant cfs-mRNA is contained in SMVs, while most cfs-miRNA is bound with protein complexes. Our data explained the stability of extracellular RNAs in human semen, and shed light on their origins and potential functions in male reproduction, and strategy of developing them as biomarkers of male reproductive system.     

作物耐热性的评价

对作物耐热性进行评价,是耐热性机制研究和耐热性品种选育的基础。在综述了作物耐热性评价的基础、评价方法以及评价指标的基础上,展望了作物耐热性评价的研究方向。     

作物耐热性的评价

对作物耐热性进行评价,是耐热性机制研究和耐热性品种选育的基础.在综述了作物耐热性评价的基础、评价方法以及评价指标的基础上,展望了作物耐热性评价的研究方向.     

mRNA and microRNA quality control for RT-qPCR analysis

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like RT-qPCR and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency.RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of RT-qPCR as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity.     

植物耐热性及热激信号转导机制研究进展

随着温室效应的加剧,全球气候变暖已经成为现代农业生产体系所面临的严峻挑战．高温灾害性气候是影响作物产量的一种主要的非生物胁迫．因此,对于农作物生产而言,研究植物耐热信号转导机制不仅有重要的科学意义,而且有现实的紧迫性．最近几年,在阐明植物耐热信号转导机制的研究方面取得了很多重要的进展,这些进展涵盖植物高温胁迫的感受机制、热激转录因子和热激蛋白的表达调控、热激转录因子结合蛋白参与耐热性调控的分子机制等几个主要的方面．热胁迫影响细胞膜系统、RNA、蛋白质的稳定性,同时改变酶的活性和细胞骨架系统．当热胁迫来临时,植物的转录组会发生显著变化,所涉及的基因大约占基因组的2％．这些高温胁迫响应基因构成了热激响应网络,是植物抵御热胁迫的第一道防线．植物的耐热性分为基础耐热性和获得性耐热性．基础耐热性是植物固有的耐热性．获得性耐热性是温和的热驯化诱导的耐热性．获得性耐热性状的形成反映了植物在自然生长环境下适应高温胁迫的生理机制．     

薄芝糖肽对小鼠T细胞IL-2、IL-3 mRNA表达的影响

目的:探讨薄芝糖肽对小鼠T细胞IL-2、IL-3 mRNA表达的影响.方法:制备静息T淋巴细胞和活化T淋巴细胞,收集细胞并提取总mRNA,进行RT-PCR扩增,测定IL-2活性和IL-3活性,并对数据进行t测验.结果:小鼠IL-2、IL-3 mRNA表达量随薄芝糖肽浓度的增加而升高,且有一定的浓度依赖性.结论:薄芝糖肽免疫增强和抗肿瘤作用的基础是与其在转录水平增强IL-2 mRNA的表达分不开的.T细胞是薄芝糖肽作用的靶细胞,而且说明了增强T细胞中两种细胞因子mRNA的表达是薄芝糖肽免疫调节的重要作用机制.