Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels ¹. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest ². As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens ³. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources ^4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure ¹². Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.     

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC. Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.     

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.     

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.     

示差扣除杂交法及其在分子生物学中的应用

对示差扣除杂交方法对中人们比较熟悉的示差筛选,扣除杂交和ＤＤＲＴ－ＰＣＲ法,以及近几年新提出的ＲＤＡ、ＳＳＨ和ＲＳＤＤ法等六种主要的差异基因筛选法,从其原理,在分子生物学中的应用,优缺点及其改进进行了简要概述。     

Diagnosis of Dermatophytosis Using Molecular Biology

Identification of fungi in dermatological samples using PCR is reliable and provides significantly improved results in comparison with cultures. It is possible to identify the infectious agent when negative results are obtained from cultures. In addition, identification of the infectious agent can be obtained in 1 day. Conventional and real-time PCR methods used for direct fungus identification in collected samples vary by DNA extraction methods, targeted DNA and primers, and the way of analysing the PCR products. The choice of a unique method in a laboratory is complicated because the results expected from skin and hair sample analysis are different from those expected in cases of onychomycosis. In skin and hair samples, one dermatophyte among about a dozen possible species has to be identified. In onychomycosis, the infectious agents are mainly Trichophyton rubrum and, to a lesser extent, Trichophyton interdigitale, but also moulds insensitive to oral treatments used for dermatophytes, which renders fungal identification mandatory. The benefits obtained with the use of PCR methods for routine analysis of dermatological samples have to be put in balance with the relative importance of getting a result in a short time, the price of molecular biology reagents and equipment, and especially the time spent conducting laboratory manipulations.     

原位PCR和原位杂交检测蛋鸡J亚群禽白血病病毒

根据ALV_J原型株HPRS10 3株gp85基因的内部序列 ,和pol基因的 3′端设计一对引物H5 H7。从发生ML病死蛋用型鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取总RNA ,反转录为cDNA ,经PCR扩增得到长度为 5 4 5bp的ALV_JcDNA特异性探针。探针定位于 5 2 5 8～ 5 80 2bp。将病鸡的组织石蜡切片置HybaidExpress原位PCR仪平台上 ,以H5 H7为引物进行原位PCR扩增。应用地高辛标记的cDNA探针对原位PCR扩增后切片进行了原位杂交检测。结果在待检组织肿瘤组织、十二指肠、骨髓中出现明显的阳性信号。睾丸、肺、胰腺、大脑、输卵管、肾脏均检出散在的阳性信号。这是国内外首次从分子水平证明蛋鸡J亚群禽白血病。     

斑马鱼整体原位杂交的技术改良

斑马鱼整体原位杂交技术广泛应用于基因表达谱、基因间相互关系和突变体筛选等方面,是研究斑马鱼发育相关基因功能的重要技术。本文从杂交探针的制备、浓度的选择和洗脱以及胚胎的脱色、蛋白酶K消化、底物显色等方面进行了摸索、改进及简化,获得了背景低、着色清晰、特异性高的实验结果,预示了简单实用、成本低廉的斑马鱼整胚原位杂交技术平台的成功建立。     

History in the Gene: Negotiations Between Molecular and Organismal Anthropology

In the advertising discourse of human genetic database projects, of genetic ancestry tracing companies, and in popular books on anthropological genetics, what I refer to as the anthropological gene and genome appear as documents of human history, by far surpassing the written record and oral history in scope and accuracy as archives of our past. How did macromolecules become “documents of human evolutionary history”? Historically, molecular anthropology, a term introduced by Emile Zuckerkandl in 1962 to characterize the study of primate phylogeny and human evolution on the molecular level, asserted its claim to the privilege of interpretation regarding hominoid, hominid, and human phylogeny and evolution vis-à-vis other historical sciences such as evolutionary biology, physical anthropology, and paleoanthropology. This process will be discussed on the basis of three key conferences on primate classification and evolution that brought together exponents of the respective fields and that were held in approximately ten-years intervals between the early 1960s and the 1980s. I show how the anthropological gene and genome gained their status as the most fundamental, clean, and direct records of historical information, and how the prioritizing of these epistemic objects was part of a complex involving the objectivity of numbers, logic, and mathematics, the objectivity of machines and instruments, and the objectivity seen to reside in the epistemic objects themselves.     

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell ¹.Open in a separate windowClick here to view.^{(64M, flv)}     

The Biology of Prostaglandins and Related Eicosanoids in Invertebrates: Cellular, Organismal and Ecological Actions

SYNOPSIS. Prostaglandins and related eicosanoids are oxygenatedmetabolites of C20 polyunsaturated fatty acids. These compoundshave been detected in species representing all major animalphyla. The significance of eicosanoids lies in two broad areasof animal biology. In one, eicosanoids are involved in regulationof many cellular events. In the other, eicosanoids facilitatecertain ecological interactions. Eicosanoids are known bestin the narrow context of their clinical signif-icance in humanmedicine. In this essay we suggest a new, broader paradigm forunderstanding the meaning of eicosanoids. Under this paradigm,called the biological paradigm, we note eicosanoids were recruitedinto roles as biological signal moieties long before the originsof the Metazoa. During the ensuing evolutionary diversificationof animals, eicosanoids have been used in a vast diversity ofbiolog ical roles, some of which occur only in invertebrates.We think this diversity endows eicosanoids with unusual explanatorypower in apprehending biological phenomena. In this review,we recount the literature on eicosanoids in protozoans and procaryotes,then provide a detailed review of the roles of eicosanoids ininverte-brate immunity. We draw upon recent work in parasitoiogyto outline an ecological role of eicosanoids in host-parasiterelationships. It appears to us that eicosanoids exert profoundeffects at the cellular, organismal and ecological levels ofbiologicalorganization. We suggest that continued inquiry into the biologicalsignificance of eicosanoids will yield important new informationon invertebrates.     

荧光原位杂交技术的研究进展

荧光原位杂交(FISH)是在染色体、间期细胞核和DNA纤维上进行DNA序列定位的一种有效手段。近年来,围绕提高检测的分辨率和灵敏性,不断将免疫染色、量子点和微流控芯片等物理化学技术引入到荧光原位杂交中,促进了它的快速发展。本文主要综述了荧光原位杂交的基本原理和发展历程,重点介绍了免疫染色-荧光原位杂交(immuno-FISH)、量子点-荧光原位杂交(QD-FISH)和微流控芯片-荧光原位杂交(FISH on microchip)等多种新技术及其检测特点,如快速、灵敏、动态、多样化等。随着荧光原位杂交技术的不断完善与发展,将在细胞遗传学、表观遗传学及分子生物学等领域发挥更加重要的作用。