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原位杂交技术的一些应用技巧

目的:检测基因在动物组织或细胞中的时空表达模式。方法:转录反义RNA探针;利用RNA原位杂交技术检测人和小鼠牙原基中若干基因的表达。结果与结论:通过优化条件,转录出完整的反义RNA探针,并成功地利用RNA原位杂交技术在组织中检测到基因的表达;分析了一些在RNA原位杂交的过程中可能碰到的问题及其解决方法。     

原位杂交技术在植物遗传育种上的应用

本文在简要介绍原位杂交技术的基础之上,重点介绍了该技术在植物遗传育种领域,即在(1)异源染色质及染色体畸变检测;(2)植物基因工程及基因表达研究;(3)构建植物基因物理图谱;(4)染色体RNA研究等方面的应用现状,并对原位杂交技术在提高检出率,与染色体显带技术结合,PCR-原位杂交等方面提了一些见解。     

原位杂交技术在植物遗传育种上的应用

本文在简要介绍原位杂交技术的基础之上 ,重点介绍了该技术在植物遗传育种领域 ,即在 (1 )异源染色质及染色体畸变检测 ;(2 )植物基因工程及基因表达研究 ;(3)构建植物基因物理图谱 ;(4)染色体RNA研究等方面的应用现状 ,并对原位杂交技术在提高检出率 ,与染色体显带技术结合 ,PCR 原位杂交等方面提了一些见解。     

斑马鱼高分辨率整胚原位杂交实验方法与流程

整胚原位杂交技术是利用反义RNA探针检测体内mRNA表达的一项技术, 在利用模式动物研究基因时空表达方面有着重要的应用。如何使用该技术得到特异、高敏感度的表达结果, 对每一个使用该技术的实验室来说都很重要。本实验室参照常规的实验方法, 对该技术加以改进, 使之更加灵敏, 结果更加特异。文章主要以斑马鱼为例, 介绍了整胚原位杂交技术的发展历史, 并重点介绍了本实验室所用的整胚原位杂交实验流程, 同时还分析了实验结果不理想的原因及其解决方法。     

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…     

以地戈辛标记反义RNA为探针的组织原位杂交

原位分子杂交是在液相和固相分子杂交基础上发展起来的,从染色体、细胞或整体水平在原位研究特异核酸的分布、数量、表达形态及其同细胞的分化、生理或病理状态、形态特征演化之间关系的分子生物学技术。随着技术的发展,原位杂交不仅被用来研究特定DNA、RNA的细胞内的分布,而且被用来探索被检mRNA相关基因的表达。可以说,原位杂交技术在分子生物学与形态学研究之间架起了一座桥梁。     

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...     

神经坏死病毒在赤点石斑鱼组织中的分布

研究采用地高辛原位杂交和免疫荧光检测技术分别检测病毒RNA2和衣壳蛋白在患病赤点石斑鱼Epinephelus akaara稚鱼中分布。地高辛检测结果表明病毒RNA2主要分布在脑、脊髓、视网膜和鳃上; 免疫荧光检测结果和地高辛检测结果一致, 表明病毒靶器官主要也是脑、脊髓、视网膜和鳃。肠道中几乎检测不到阳性信号, 可能不是病毒的靶器官。因此可以推测神经坏死病毒感染赤点石斑鱼的主要途径是鳃, 而不是肠道。       

J Clin Cancer Res：研究识别早期乳腺癌免疫治疗的新靶标

近日,耶鲁大学癌症中心研究人员利用一种新的分子分析工具RNAScope。准确地检测出针对早期乳腺癌的免疫治疗的一个重要靶标的水平。诊断测试使用RNAScope,测量福尔马林固定的癌组织中PD—LI（细胞程序性死亡配体PDL1）mRNA的量。RNAscope是一种新型RNA原位杂交技术。基于其独特的探针设计与背景抑制技术,并且融合传统RNA原住杂交技术与FISH技术的优点,使单个RNA的转录变得可视化,是目前最精准的RNA检测技术。PD—L1是目前几种新型免疫刺激疗法的靶标。这项研究结果发表在Clinical Cancer Research杂志上。     

Highly sensitive fluorescent-labeled probes and glass slide hybridization for the detection of plant RNA viruses and a viroid

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing ~(32)p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a ~(32)P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.     

mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes

Recently, we have established an in-tube in situ hybridization method named mRNA quantification after fluorescence activated cell sorting (FACS-mQ), in which a specific RNA in a particular cell type is stained with a florescent dye, allowing the stained cells to be selected by FACS without suffering excessive RNA degradation. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we used locked nucleic acid (LNA) oligonucleotides, which are known to enhance both the sensitivity and specificity of RNA detection, as hybridization probes in FACS-mQ. When we used a LNA probe targeting the human 28S sequence, we were able to efficiently separate human cells from rat cells. Using LNA probes, the hybridization step was shortened to 1 h. After the hybridization step, 84.6% RNA was preserved; thus, we were able to successfully measure gene expression levels in each type of cell after FACS. Providing the LNA probe efficiently hybridizes with the target sequence, FACS-mQ with an LNA probe is a powerful tool for separating particular cells and determining their biological characteristics by analyzing their gene expression profile.     

Ribosomal DNA sequences detected in malaria parasites by cytochemical hybridization

A procedure in which fluorochrome-labelled RNA is hybridized in situ to homologous DNA sequences was used to investigate the possible application of this method to the human malaria parasite Plasmodium falciparum. Rhodamine-labelled ribosomal RNA stained the nuclei of the parasites after cytochemical hybridization. This result demonstrates that ribosomal RNA genes can be visualised. It was estimated that the hybridization efficiency was greater than 40%.     

Ribosomal DNA sequences detected in malaria parasites by cytochemical hybridization

A procedure in which fluorochrome-labelled RNA is hybridized in situ to homologous DNA sequences was used to investigate the possible application of this method to the human malaria parasite Plasmodium falciparum. Rhodamine-labelled ribosomal RNA stained the nuclei of the parasites after cytochemical hybridization. This result demonstrates that ribosomal RNA genes can be visualised. It was estimated that the hybridization efficiency was greater than 40%.     

Quantitation of labeled globin messenger RNA by hybridization with excess complementary DNA covalently bound to cellulose.

A method is described to quantitate labeled globin mRNA by hybridization with excess cDNA which was enzymatically polymerized on oligo(dT)-cellulose. In a large excess of cDNA-cellulose the rate of RNA hybridization was dependent on DNA concentration and not on RNA concentration. Nonhybridized RNA can be digested by RNase and washed from the cDNA which is covalently bound to cellulose. This enables the detection of labeled globin mRNA even when present in a porportion as low as 0.02-0.03% of the total RNA.     

One-hour downward alkaline capillary transfer for blotting of DNA and RNA.

The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.