Application of restriction display PCR technique in the preparation of cDNA microarray probes

AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes. METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template. The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis. RESULTS: A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced, which were the specific gene fragments of HCV. These fragments could be further used as probes in microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility, and linearity in detecting HCV RNA were satisfactory. CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.     

蛋白芯片在寄生虫病免疫学诊断中的应用前景

蛋白芯片技术是近年来兴起的一种高度集成化的分析和研究手段,该技术具有高通量、快速、高效、操作简单、结果准确、灵敏度高等特点,在生命科学、临床医学、司法鉴定、卫生检验领域有很好的应用前景.该文阐述了蛋白芯片技术的基本原理、分类、制备,结合寄生虫病诊断的内容和特点,就蛋白芯片技术在寄生虫病免疫学检测领域的应用作一简要综述和展望.     

蛋白芯片在寄生虫病免疫学诊断中的应用前景

蛋白芯片技术是近年来兴起的一种高度集成化的分析和研究手段,该技术具有高通量、快速、高效、操作简单、结果准确、灵敏度高等特点,在生命科学、临床医学、司法鉴定、卫生检验领域有很好的应用前景.该文阐述了蛋白芯片技术的基本原理、分类、制备,结合寄生虫病诊断的内容和特点,就蛋白芯片技术在寄生虫病免疫学检测领域的应用作一简要综述和展望.     

Microarray-based identification of novel biomarkers in asthma.

Bronchial asthma is a complicated and diverse disorder affected by genetic and environmental factors. It is widely accepted that it is a Th2-type inflammation originating in lung and caused by inhalation of ubiquitous allergens. The complicated and diverse pathogenesis of this disease yet to be clarified. Functional genomics is the analysis of whole gene expression profiling under given condition, and microarray technology is now the most powerful tool for functional genomics. Several attempts to clarify the pathogenesis of bronchial asthma have been carried out using microarray technology, providing us some novel biomarkers for diagnosis, therapeutic targets or understanding pathogenic mechanisms of bronchial asthma. In this article, we review the outcomes of these analyses by the microarray approach as applied to this disease by focusing on the identification of novel biomarkers.     

组织芯片在胰腺癌研究中的应用现状

胰腺癌是一种早期症状隐匿、进展迅速、预后极差的恶性肿瘤。临床迫切需要寻找胰腺癌的早期诊断标记物和新的治疗靶点，以延长患者生存期。组织芯片是一种新型高通量分析技术，具有样本量大、高效、快速等特点。本文对组织芯片在胰腺癌研究，包括肿瘤标记物筛选、发病机制和预后分析、鉴别诊断、寻找治疗靶点中的应用现状作一综述。     

Difficult airway management

Airway management is unequivocally the most important responsibility of the emergency physician. No matter how prepared for the task, no matter what technologies are utilized, there will be cases that are difficult. The most important part of success in the management of a difficult airway is preparation. When the patient is encountered, it is too late to check whether appropriate equipment is available, whether a rescue plan has been in place, and what alternative strategies are available for an immediate response. The following article will review the principles of airway management with an emphasis upon preparation, strategies for preventing or avoiding difficulties, and recommended technical details that hopefully will encourage the reader to be more prepared and technically skillful in practice.     

Gene expression arrays as a tool to unravel mechanisms of normal tissue radiation injury and prediction of response

Over the past 5 years there has been a rapid increase in the use of microarray technology in the field of cancer research. The majority of studies use microarray analysis of tumor biopsies for profiling of molecular characteristics in an attempt to produce robust classifi ers for prognosis. There are now several published gene sets that have been shown to predict for aggressive forms of breast cancer, where patients are most likely to benefit from adjuvant chemotherapy and tumors most likely to develop distant metastases, or be resistant to treatment. The number of publications relating to the use of microarrays for analysis of normal tissue damage, after cancer treatment or genotoxic exposure, is much more limited. A PubMed literature search was conducted using the following keywords and combination of terms: radiation, normal tissue, microarray, gene expression profi ling, prediction. With respect to normal tissue radiation injury, microarrays have been used in three ways: (1) to generate gene signatures to identify sensitive and resistant populations (prognosis); (2) to identify sets of biomarker genes for estimating radiation exposure, either accidental or as a result of terrorist attack (diagnosis); (3) to identify genes and pathways involved in tissue response to injury (mechanistic). In this article we will review all (relevant) papers that covered our literature search criteria on microarray technology as it has been applied to normal tissue radiation biology and discuss how successful this has been in defining predisposition markers for radiation sensitivity or how it has helped us to unravel molecular mechanisms leading to acute and late tissue toxicity. We also discuss some of the problems and limitations in application and interpretation of such data.     

Bootstrapping cluster analysis: assessing the reliability of conclusions from microarray experiments

We introduce a general technique for making statistical inference from clustering tools applied to gene expression microarray data. The approach utilizes an analysis of variance model to achieve normalization and estimate differential expression of genes across multiple conditions. Statistical inference is based on the application of a randomization technique, bootstrapping. Bootstrapping has previously been used to obtain confidence intervals for estimates of differential expression for individual genes. Here we apply bootstrapping to assess the stability of results from a cluster analysis. We illustrate the technique with a publicly available data set and draw conclusions about the reliability of clustering results in light of variation in the data. The bootstrapping procedure relies on experimental replication. We discuss the implications of replication and good design in microarray experiments.     

鼠疫诊断芯片的探针制备

目的 探索制备鼠疫诊断芯片特异性探针的新方法。方法 直接采用限制性核酸内切酶Sau3A Ⅰ消化鼠疫耶尔森菌3个致病性质粒，克隆在T载体上，再经巢式PCR扩增制备成打印芯片的探针。结果 该法收集到250条探针用于打印芯片。分别与鼠疫耶尔森菌，同属的假结核耶尔森菌和小肠结肠炎耶尔森菌杂交，可筛选出杂交信号强阳性的特异点阵。结论 该法简便可行，适于制备鼠疫诊断芯片的探针。     

Changes in BCG strains

BCG originated from a virulent bovine strain of the tubercle bacillus after prolonged serial subculture on a potato medium. Since attenuation was achieved, the BCG strain has been distributed to a large number of centres where BCG vaccine is produced. Many of these production laboratories have maintained their BCG lines by continuing serial transfers, but have employed a variety of media for this purpose, and have produced BCG vaccine by a variety of techniques. Distinct differences have developed between some of the daughter strains of BCG, but the mechanism through which these changes have occurred has not been clear. In recent years methods have been developed which have enabled changes taking place within some BCG strains during experimental serial subculture to be monitored. In this survey the relationship of the changes observed to the different techniques employed for the maintenance of BCG lines and for the preparation of vaccine is considered. It is suggested that selection of minority populations within BCG strains noted during experimental studies may provide an analogy with the mechanism through which the original attenuation of the virulent bovine strain was brought about. The relevance of small-scale laboratory investigations to full-scale production procedures is also discussed, and finally some additional measures that might be taken to minimise changes in BCG strains are proposed.     

Microarray analysis of evolution of RNA viruses: evidence of circulation of virulent highly divergent vaccine-derived polioviruses

Two approaches based on hybridization of viral probes with oligonucleotide microarrays were developed for rapid analysis of genetic variations during microevolution of RNA viruses. Microarray analysis of viral recombination and microarray for resequencing and heterogeneity analysis were able to generate instant genetic maps of vaccine-derived polioviruses (VDPVs) and reveal the degree of their evolutionary divergence. Unlike conventional methods based on cDNA sequencing and restriction fragment length polymorphism, the microarray approaches are better suited for analysis of heterogeneous populations and mixtures of different strains. The microarray hybridization profile is very sensitive to the cumulative presence of small quantities of different mutations, including those that cannot be revealed by sequencing, making this approach useful for characterization of profiles of nucleotide sequence diversity in viral populations. By using these methods, we identified a type-3 VDPV isolated from a healthy person and missed by conventional methods of screening. The mutational profile of the polio strain was consistent with >1 yr of circulation in human population and was highly virulent in transgenic mice, confirming the ability of VDPV to persist in communities despite high levels of immunity. The proposed methods for fine genotyping of heterogeneous viral populations can also have utility for a variety of other applications in studies of genetic changes in viruses, bacteria, and genes of higher organisms.     

采用人基因表达谱芯片筛选肺癌转移相关基因的初步研究

目的应用基因芯片技术分析人高、低转移肺巨细胞癌细胞株的基因差异表达谱，筛选与肺癌转移相关基因。方法提取人高转移肺巨细胞株95D和人低转移肺巨细胞株95C的mRNA，通过逆转录，制备分别用cy5-dUTP和cy3-dUTP进行标记的cDNA探针，并与芯片杂交。经过ScanArray3000扫描仪扫描，获取图象及对杂交信号进行数据分析，筛选出95D和95C细胞表达差异的基因谱，并对其中部分基因进行RT—PCR分析、验证。结果在所检测95C和95D细胞中，466条基因有表达差异，其中具有同-Genebank量的有108对，上调基因47对，下调基因61对。对其中KIAA1108、PGR1、JWA、S182、Jab1部分差异表达基因，经RT-PCR技术验证，结果与基因芯片基本符合。结论肺癌转移过程与多种基因作用有关，基因芯片技术可为肺癌转移相关基因的筛选，提供有效方法。     

Population Screening for Hemoglobinopathy Profiling: Is the Development of a Microarray Worthwhile?

In order to perform affordable and expedient whole population scans for the single nucleotide polymorphisms (SNPs) involved in hemoglobinopathies, microarrays based on single nucleotide extension (SNE) might prove advantageous to whole genome/exome sequencing in terms of cost, speed, interpretation and discretion as they focus on a very small part of the tested genome. The development of a microarray assay entails most of the cost, to be deferred by the massive use of the end product. A microarray assay development project, involving multiplex polymerase chain reaction (PCR), labeling, hybridization and scanning/scoring steps is presented as a paradigm of objective bug ratios expected to such procedures and of ways to cope with them. Qualification of the microarray genotypes needs a reference method, which may still be restriction digestion or other, as sequencing remains an expensive commodity. Optimization of wet steps should also be followed by careful and perhaps individualized dye excitation and in silico scoring rules, taking into consideration decay and bleaching effects that perplex development. The strategy of successive elimination of problems, a top-bottom procedure, which had been used and is usually preferred by developing agencies, might have been erroneous; a bottom-up course to delineate issues in different levels, although more laborious, might be the correct choice, especially as software and robotic hardware, high throughput tools become more mature and available. The testing for interlocus compatibility, specificity and robustness is demanding and warranted only in the case of steady, high volume use of an assay for territorial, national or international use.     

Colonoscopy 'my way': preparation, anticoagulants, antibiotics and sedation.

Colonoscopy was introduced in the 1960s. The facility with which this technique is performed has been enhanced by vast improvements in instrumentation. In spite of this, physician attitudes concerning colonoscopy have changed little over the past several decades. The diet for precolonoscopic preparation has not been altered for 30 years. Colonoscopists have a great reluctance to use a new preparation instead of the 4 L electrolyte solution, perhaps because this was such a significant advance in colonoscopic cleansing, its predecessor being castor oil and enemas. Physicians continue to be wary of the patient who is taking acetylsalicylic acid in the absence of any studies that show that this is detrimental for polypectomy. The management of the patient on warfarin anticoagulation remains a subject for debate. As for antibiotic prophylaxis, most endoscopy units do not have a standardized approach, although there are good guidelines that, if followed, should decrease the risk of infective endocarditis. Sedation for the endoscopic examination is usually administered by the colonoscopist, although anesthesiologists may, in some countries (and in some defined areas of the United States) be the primary administrators of sedation and analgesia. The present article is a personal approach to the following issues: the preparation of the colon for an examination, current thoughts about anticoagulation and acetylsalicylic acid, antibiotic prophylaxis for colonoscopy and the technique for sedation out of the hospital.     

应用基因芯片技术研究甲状腺乳头状癌的基因表达

目的　应用基因芯片技术研究甲状腺乳头状癌 (PTC)和正常成人甲状腺组织基因的差异表达。方法　分别用Cy5和Cy3两种不同的荧光染料通过逆转录反应将PTC组和对照组甲状腺组织的mRNA分别标记成探针 ,并与载有一组靶基因的基因表达谱芯片进行杂交。通过扫描荧光强度 ,计算机软件分析 ,寻找两组差异表达基因 ,并用RT PCR、免疫组化对其中两条基因进行验证。结果　共有 65条差异表达基因 ,其中表达增加的有 48条 (2 .0倍以上 ) ,表达降低的有 17条 (0 .5倍以下 )。RT PCR、免疫组化结果与芯片扫描结果一致。结论　基因芯片是筛选PTC与正常成人甲状腺组织差异表达基因的有效方法。通过筛选所得差异基因提示 ,PTC的发病涉及细胞外基质、细胞因子、受体信号转导等多个方面。     

Microarray Databases I

Microarray analyses, in particular for pancreatic cancer and pancreatitis, have been extensively performed. Today, these data are so extensive that meta-analyses of microarray experiments for pancreatic diseases have begun to emerge. Thus, there is a large amount of data, derived from this methodology, which can help curious individuals develop, for instance, novel markers and assays of pancreatic diseases based upon the data deposited in these databases without the great expenses of generating the initial microarray experiment. Following, we provide a few sites that together constitute the first part of a small series of ‘Pancreatology and the Web’ articles on this topic.