RNA原位杂交实用技术

利用互补RNA为探针进行原位杂交是分析组织或细胞内RNA分布的行之有效的方法,通过对mRNA分布的研究可以了解特定基因的表达情况。原位杂交技术过程较长,操作繁琐,从而在一些实验中不能得到很好的使用,为此本文根据我们过去的实际操作经验对该技术中的一些使用技巧作简要的介绍。     

RNA原位杂交实用技术

利用互补RNA为探针进行原位杂交是分析组织或细胞内RNA分布的行之有效的方法 ,通过对mRNA分布的研究可以了解特定基因的表达情况。原位杂交技术过程较长 ,操作繁琐 ,从而在一些实验中不能得到很好的使用 ,为此本文根据我们过去的实际操作经验对该技术中的一些使用技巧作简要的介绍。     

细胞内RNA的原位杂交

本文介绍用原位杂交方法测定细胞内的RNA。该方法特异性较高,能保持细胞曲完整性。我们测定了不同细胞的rRNA基因的转录和原癌基因c-myc,c-H-rgs的转录,取得了较满意的结果。RNase处理细胞或质粒pBR322作探针,细胞中显影颗粒很少。本文对原位杂交方法学进行了初步的讨论。     

昆虫小器官RNA荧光原位杂交技术

【目的】在昆虫基因表达和功能研究中,RNA原位杂交技术越来越受到青睐。该技术不仅能定性定量反应基因表达的时空特异性,而且能在细胞水平上检测基因表达的调控模式。为了将该技术更好地在昆虫小器官研究中运用,我们以果蝇幼虫翅芽为例优化了改技术。【方法】解剖果蝇3龄幼虫翅芽进行原位杂交实验。【结果】我们发现影响原位杂交结果的因素十分复杂,包括取材时期,探针的合成,预杂交/杂交的时间和温度,清洗时间,适当的对照等。通过RNA荧光原位杂交实验,我们揭示了调控细胞记忆的trithorax基因在3龄翅芽广泛表达,并且受到转录因子Optomotor-blind的负调控。【结论】这一技术方法为研究昆虫小器官的基因表达和调控提供了便捷手段。     

RNA原位杂交技术及其在植物基因表达研究中的应用

原位杂交 ( In situ Hybridization)是一种在细胞水平上研究基因表达调控的最直接有效的分子生物学技术。这一技术最初应用于动物染色体上的基因物理定位 〔1〕和特定 m RNA在组织中的空间定位〔2〕,后来又作为诊断工具检测感染病毒的细胞 〔3〕。到 80年代后期 ,原位杂交技术开始应用于植物基因表达调控的研究 〔4～ 6〕。植物基因的时空表达研究是探讨植物生长发育机制的重要手段。由于 RNA原位杂交技术能够精确确定基因表达的时空分布 ,而得到了越来越广泛的应用 ;从营养器官生长发育〔7～ 9〕、生殖器官生长发育〔10～ 13〕、自交不…     

植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...     

骨组织总RNA的提取技术

目的探讨Trizol试剂提取骨组织总RNA的技术方法。方法采用高速均质仪进行组织粉碎处理,用Tr-izol试剂提取RNA后,分3组进行RNA纯化处理,并通过紫外分光光度计、电泳、Real-time PCR等方法验证RNA的质量。结果用Tirzol试剂从经粉碎处理的骨组织中所提取的总RNA纯度差,只有再进行DNase1消化及有机溶剂再纯化后,才能保证所提RNA完整且纯净。结论从组织中提取的总RNA经DNase1消化及有机溶剂抽提,可提高RNA质量;另外实验证实用紫外分光光度法判断RNA的质量有许多局限性,利用琼脂糖电泳及Real-time PCR方法也能很简便的判断RNA质量。     

以反意义RNA为探针的原位杂交组织化学方法

原位杂交组织化学方法是在组织及细胞水平上研究基因表达及调控的重要方法之一。我们利用一个由体外转录产生的与小鼠阿黑促皮原(POMC)mRNA顺序互补的反意义RNA为探针,直接在大鼠垂体的组织切片上进行杂交反应。结果表明,杂交在反意义RNA探针及POMC mRNA之间进行,并形成稳定的杂交分子。放射自显影影像显示出垂体中POMC mRNA的分布及相对含量。     

植物基因表达的组织原位杂交和原位组织化学分析技术的改进

改良了组织原位杂交和原位酶组织化学分析的方法。主要改进有：原位杂交采用简化的ＦＡＡ固定程序,常规石蜡包埋,切片时采取液氮深冷冻;以随机引物法标记的ＤＮＡ代替转录标记的ＲＮＡ作探针,在保湿盒中进行杂交,而不用矿物油覆盖。组织化学分析中用含铁氰化钾的显色液进行ＧＵＳ染色后再包埋、切片的程序,代替先包埋、切片再显色的常规方法,可最大限度地保持酶活性的真实性。     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

A Comparison of Digoxigenin and Biotin Labelled DNA and RNA Probes for in Situ Hybridization

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes acheived maximal sensitivity without use of biotin-streptavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.     

Human Papillomavirus Detection In Cervical Cells By In Situ Hybridization With Biotinylated Probes

We have investigated the applicability of human papillomavirus (HPV) DNA detection by in situ hybridization with biotinylated probes in epithelial cells obtained from the cervix using a cotton tip swab. We describe a simple procedure for obtaining homogeneous cell samples and good preservation of cellular structure. This is achieved by pretreatment of cells with L-cysteine before hybridization. Separate denaturation of cellular DNA and probe DNA is also necessary for satisfactory results. Both benign HPV DNA 6/11 and potentially oncogenic HPV DNA 16/18 could be identified in our series. In situ hybridization on cervical scrapes is a rapid, simple and very specific method for detecting patients infected with oncogenic HPV types.     

Detection of Bacteria in Phagocyte-Smears from Septicemia-Suspected Blood by In Situ Hybridization Using Biotinylated Probes

We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.     

体外诱导分化神经细胞的原位分子杂交方法

经１×１０－６ｍｏｌ／Ｌ视黄酸诱导的Ｐ１９细胞体外可向神经方向分化,接种于多聚赖氨酸（ｐｏｌｙＤｌｙｓｉｎｅ）和纤连蛋白（ｆｉｂｒｏｎｅｃｔｉｎ）包被的玻片后,细胞逐渐聚集成团,此时细胞的贴壁性较差,进行原位分子杂交时容易脱落。我们尝试在细胞表面覆盖一层明胶,减少了细胞的脱落,又比较了蛋白酶Ｋ和胃蛋白酶对细胞蛋白质的消化作用,确定胃蛋白酶可较温和地消化细胞蛋白质,使探针有效地透入结合,杂交后细胞亦能较完整地保留于玻片上。     

原位杂交技术及其在果树研究中的应用

原位杂交技术是近年来快速发展起来的一门新技术,本文介绍了原位杂交技术的基本原理、方法及其发展前景,以及该技术与其它生物学技术相结合而形成的一些新技术。综述了这些技术在果树研究中的应用情况。     

In Situ Hybridization of Lilium Whole Mount Synaptonemal Complex Chromosomal Preparations

Whole mount meiotic preparations of the synaptonemal complex complement of Lilium have been used for in situ hybridization experiments. A probe of the maize ribosomal DNA gene cluster has been successfully hybridized to the lily preparations. Three strong signals, corresponding to the three known lily nucleolus organizer regions, have been seen in most of the chromosome preparations. In situ hybridization experiments using meiotic preparations should be useful for identifying specific chromosomes, and for investigating the role of particular DNA molecules important to meiotic function.     

生物素标记的人IL-6受体基因探针的制备及在细胞原位杂交中的应用

制备了人IL-6受体cDNA 1.7 kb片段及其胞外区近膜侧区段0.5kb片段,分别用生物素标记制备了人IL-6受体基因探针,用斑点杂交分析了探针的灵敏度和特异性,并将探针用于四种白血病细胞系的原位杂交。结果表明,探针的灵敏度可达15～125pg/μl、特异性较好,所检测的四种细胞的IL-6受体mRNA表达水平与其细胞膜表面IL-6受体表达水平相一致。     

应用小鼠整体原位杂交技术检测MDM2在鼠胚发育不同阶段的表达

整体原位杂交(whole-mountinsituhybridization,WMH)已经成为基因表达定位和表达分布模式研究的一种重要手段.该技术能在整体水平上精确地研究胚胎发育过程中基因表达的三维信息,而且为大规模筛选区域及组织特异性候选克隆提供了有利的技术手段.采用体外转录地高辛标记的RNA探针,检测已知基因MDM2在鼠胚胎发育不同阶段的表达模式.