A novel method for constructing murine cDNA library enriched with maternal mRNAs exhibiting de novo independent post-fertilization polyadenylation

Recently, mouse maternal mRNAs such as SSEC-D, Spin, beta-catenin, Ptp4a1, and Maid have been found to exhibit de novo independent polyadenylation after fertilization. To obtain an overall picture of post-fertilization polyadenylation events, we developed a novel method for constructing murine fertilized egg cDNA library enriched with cDNAs exhibiting de novo independent polyadenylation. As a pilot study, we isolated at least four new maternal mRNAs exhibiting extension of poly(A) tail in fertilized 1-cell eggs. Moreover, various types of polyadenylation of maternal RNAs were observed at this stage, suggesting the presence of novel mechanisms for regulating the length of poly(A) tails of maternal mRNA. This is the first report of successful construction of a cDNA library enriched with newly polyadenylated maternal mRNAs derived from post-fertilized mouse eggs. This cDNA library will be useful for molecular analysis of the mechanisms underlying post-fertilization polyadenylation of mammalian maternal RNAs.     

文昌鱼性腺差减cDNA文库的构建

本研究以日本文昌鱼(Branchiostoma japonicum)性腺cDNA为材料,应用抑制性差减杂交技术构建文昌鱼雌雄性腺的差减cDNA文库.正向差减杂交以卵巢为试验方、精巢为驱动方,反向差减杂交以精巢为试验方、卵巢为驱动方,将所获差减cDNA片段克隆插入质粒表达载体,转化大肠杆菌DH5α,最后获得的正、反向差减文库分别含459、243个重组子.PCR 扩增鉴定正、反向差减cDNA文库的插入片段,其中90%左右的克隆皆能扩增出有效产物,插入片段范围为200-600 bp,符合差减文库PCR产物片段的大小.随机选取30个阳性克隆测序分析,得到26有效基因片段,进一步从中选取16个序列,用实时定量PCR对文库质量进行验证,结果表明所建文库能够达到富集雌雄性腺差异表达基因的目的.文昌鱼性腺差减文库的构建,为进一步分离、鉴定性腺分化和发育相关基因奠定了基础[动物学报 54(3):482-48 8,2008].     

标化消减杂交:提高差异表达cDNA筛选效率的新策略

在回顾分析了消减杂交技术改进历程的基础上,我们提出了“标化消碱杂交策略”,此策略的核心是“用标化cDNA制备消碱杂交反应的单链驱动子和单链示踪子,分离回收消减杂交后的单链示踪子用于制备差异表达cDNA库,并从标化cDNA库制备探讨来对差异表达cDNA库进行检测”将主要用于筛选与细胞特殊表型密切相关的（或与不同发育阶段等密切相关的）呈“全或无”差异形式表达的低丰度全长cDNA。此策略有望能较以往的消减杂交策略在降低假阳性背景、提高低丰度差异表达cDNA获得率、得到较长甚至全长差异表达cDNA等方面有较好表现,但尚待检验。     

A PCR-based method for detection and quantification of small RNAs

Recent cloning efforts have identified hundreds of thousands of small RNAs including micro RNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs). These non-coding small RNAs need to be further validated and characterized by detecting and quantifying their expression in different tissues and during different developmental courses. A simple, accurate, and sensitive method for small RNA expression profiling is in high demand. Here, we report such a PCR-based method.     

cDNA blotting offers an alternative method for gene expression studies

Northern analysis of bilberry fruit RNA using a non-radioactive detection system proved to be extremely difficult. To overcome this problem, we used cDNA instead of RNA for the blotting step. The RNA was translated to cDNA directly after isolation. The cDNA was then separated by electrophoresis, stained with ethidium bromide, and blotted onto a nylon membrane by Southern transfer. Non-radioactive detection by digoxigenin-dUTP was then possible. This method resolves several problems associated with northern blotting and is especially efficient when applied to recalcitrant plant material.     

A hybridization and enzyme blocking method to enrich minor cDNA sequences

A method is presented which allows for the enrichment of low frequency cDNA sequences. The crucial step in the procedure is the hybridization of a pool of cDNA to homologous or heterologous RNA to a Rot value which will leave minor sequences in a single strand cDNA form while the major sequences form cDNA:RNA hybrids. This allows subsequent enzymatic differentiation between major and minor sequences resulting ultimately in the degradation of the major sequences. The procedure is general and simple as it requires no column chromatography step. The method is designed to integrate into a widely used cDNA cloning protocol and results either in double-stranded cDNA which can be used for molecular cloning or as a source of probes for hybridization.     

一种提取小型昆虫总RNA的有效方法

实验采用SDS,乙酸钾(KAc)等常规化学试剂从蚜虫等小型昆虫中获得完整且纯度较高的RNA。进一步的实验证明提取的RNA可用于RT-PCR以及cDNA文库的建立等。该方法简捷、相对安全和快速、提取效率较高,适合于中小型昆虫RNA的提取。     

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.     

Improvement of an RNA purification method for grapevine (Vitis vinifera L.) suitable for cDNA library construction

A method is described, which consistently yields high quality total RNA from grapevine. Dissolving of crude RNA pellets in borate-containing buffer, instead of normally used water before a selective lithium chloride precipitation, was found to be a critical step, leading to a 2.5-fold increase of yield. The resulting RNA preparations were suitable for standard downstream applications and also for cDNA library construction. The method worked efficiently and reproducibly and could easily be scaled from milligram to gram quantities of plant material grown in hydroponic culture, sandy soil or Perlite. It was applied to different kinds of grapevine tissues (leaves, stem) and, after additional adaptation of the protocol, to roots.     

Identification of over-expressed genes in human renal cell carcinoma by combining suppression subtractive hybridization and cDNA library array

To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon membrane and the over-expressed genes were then screened by hybridizing the filter with radioactively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank database. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular endothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohistochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.     

应用限制性显示PCR技术构建细菌poly(A)化mRNA的cDNA文库

针对细菌mRNA poly(A)化位点的高度多态性,利用oligo(dT)与poly(A)特异结合的特性,以oligo(dT)一纤维素纯化mRNA,并以oligo(dT)18为引物逆转录合成cDNA,用限制性内切酶消化cDNA,所得的限制性内切酶片段与通用接头相连,通过10个选择性引物组合进行选择性PCR,使各片段得以扩增并分布于10个亚组中,并进行克隆,成功地克隆了100多个基因片段,已对其中40个进行了测序分析,探讨了限制性显示PCR技术在细菌poly(A)化mRNA cDNA库构建中的应用价值。     

Isolation and partial characterization of a novel pollen-specific cDNA with multiple polyadenylation sites from wheat

Wheat is the foremost staple food crop that offers bothcalories and proteins to a large global population. Wheat(hexaploid AABBDD genome, 16 billion bp) is a geneti-cally complex, self-pollinating plant with bisexualflowers that produce short-lived pollen. Very little is knownabout the molecular biology of its gametophyte develop-ment despite a longstanding interest in hybrid seeds. Mostof our information is from the studies on a few model andcrop plants such as Arabidopsis, tobacco, vegeta…     

Isolation of cDNA clones from an osteosarcoma-ROS17/2.8 library by differential hybridization

We have used differential hybridization to isolate and characterize two novel cDNAs expressed in chondrocytes and some osteoblastic cells. A rat osteosarcoma ROS17/2.8 cDNA library was screened and cDNA clones hybridizing strongly to radiolabeled porcine calvaria cDNA but weakly to a control radiolabeled cDNA were isolated. Two clones were obtained—p.6.1 and p.10.15. A radiolabeled probe of p10.15 was shown to hybridize specifically to a 2.3 Kb message RNA from a chondrogenic clonal cell population from rat calvaria-RCJ 3.1C5.18, and the mRNA was downregulated by 1,25 (OH)₂D₃, which inhibits chondrogenesis in these cells. The other clone, p6.1, was found to hybridize to a 0.95 Kb message that is expressed in rat liver, kidney, lung, muscle, and brain, but not expressed in spleen and expressed only in low levels in thymus.     

双链RNA病毒诱导的牙鲆胚胎细胞差减cDNA文库的构建

以紫外线灭活的dsRNA病毒草鱼出血病病毒(GCHV)诱导和模拟诱导的牙鲆胚胎细胞为材料,利用抑制性差减杂交(SSH)技术,成功构建了双链RNA病毒诱导的牙鲆胚胎细胞(FEC)差减cDNA文库.以管家基因α-tublin作为差减指标,经检测,该文库差减效率达2 10倍,表明经病毒诱导后某些差异表达基因也得到了相应倍数的富集.将获得的cDNA片段连接到pGEM-T载体,PCR检测显示差减片段在250bp～2 000bp之间.该差减cDNA文库的构建为从分子水平研究牙鲆培养细胞对dsRNA病毒的免疫反应、以及进一步鉴定和克隆差异表达基因打下了坚实基础.     

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNA later at 4 degrees C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.     

Mammalian RNA virus-derived small RNA: biogenesis and functional activity

The role of virus-derived small RNAs (vsRNAs) has been identified as an antiviral mechanism in plants, arthropods, and nematodes. Although mammalian DNA viruses have been observed to encode functional miRNAs, whether RNA virus infection generates functional vsRNAs remains under discussion. This article reviews the most recent reports regarding pathways for generating vsRNAs and the identified vsRNA activity in mammalian cells infected with RNA viruses. We also discuss several hypotheses regarding the roles of mammalian vsRNAs and comment on the potential directions for this research field.