Spotted long oligonucleotide arrays for human gene expression analysis

DNA microarrays produced by deposition (or 'spotting')of a single long oligonucleotide probe for each gene may be an attractive alternative to other types of arrays. We produced spotted oligonucleotide arrays using two large collections of approximately 70-mer probes, and used these arrays to analyze gene expression in two dissimilar human RNA samples. These samples were also analyzed using arrays produced by in situ synthesis of sets of multiple short (25-mer) oligonucleotides for each gene (Affymetrix GeneChips). We compared expression measurements for 7344 genes that were represented in both long oligonucleotide probe collections and the in situ-synthesized 25-mer arrays. We found strong correlations (r = 0.8-0.9) between relative gene expression measurements made with spotted long oligonucleotide probes and in situ-synthesized 25-mer probe sets. Spotted long oligonucleotide arrays were suitable for use with both unamplified cDNA and amplified RNA targets, and are a cost-effective alternative for many functional genomics applications. Most previously reported evaluations of microarray technologies have focused on expression measurements made on a relatively small number of genes. The approach described here involves far more gene expression measurements and provides a useful method for comparing existing and emerging techniques for genome-scale expression analysis.     

不同样本测序前处理方案对冠状病毒基因组高通量测序结果的影响

目的以基因组最大RNA病毒(冠状病毒)为代表,研究不同测序前样本处理模式对高通量测序获得病毒全基因组序列信息质量的影响。方法以细胞培养的人冠状病毒HCoV-OC43样本为代表,分为4种测序前样本处理模式,即:未处理组、核酸提取前DNase和RNase处理组、核酸提取后DNase处理组、核酸提取前DNase和RNase处理且核酸提取后DNase处理组。不同模式处理后的核酸分为两份,一份直接RNA测序(未扩增),另一份经序列非依赖的单引物扩增(SISPA)后DNA测序。结果尽管不同处理方式下获得的病毒基因组覆盖率差别不大,但是样本核酸提取后经DNase处理组直接测序获得了最高的基因覆盖度和测序准确性,而SISPA扩增可有效提高病毒测序读长(reads)比例与基因组各位点的测序深度。结论本研究为优化冠状病毒等RNA病毒全基因组测序策略提供了技术参考。     

一种用于微阵列分析的通用引物U2联合标记方法

目的报告一种新的用于微阵列研究的荧光标记技术：通用引物U2联合标记技术(UPL);比较UPL与随机引物等其他标记方法的效率和可重复性。方法分别用4种标记方法标记流感病毒RNA样品,与流感病毒寡核苷酸检测芯片杂交后,用Spss10．0软件对杂交结果进行分析。结果UPL方法在标记物杂交的荧光强度、信噪比、探针真阳性率和可重复性等方面均高于随机引物逆转录掺入标记法,而与其他两种RD标记方法相当,但标记过程相对更加简单。结论UPL方法是一种新颖而有效的标记方法,在微阵列技术研究方面具有广泛的应用价值。     

基因芯片技术检测鉴定临床常见致病真菌的初步研究

目的为了快速、简便、高通量地鉴定临床常见致病真菌,建立了一种采用基因芯片技术对临床常见的致病真菌鉴定的分子生物学方法。方法以5.8S rDNA与28S rDNA间的内转录间区2(internal transcribed spacer-2,ITS-2)为靶标,针对待检的临床常见致病真菌设计合成一系列寡核苷酸探针,制成寡核苷酸芯片。待检真菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱分析归纳,得到一套种特异性的典型杂交图谱。待检的样品菌与基因芯片杂交,得到的杂交结果与典型图谱比对即可判断出样品的种类。结果以涉及8个属20个种的标准致病真菌菌株对芯片的特异性、重复性、灵敏度进行考察,结果表明,该研究建立的基因芯片技术可以有效地区分20种临床常见致病真菌,特异性良好,重复性良好(信噪比CV<10%),灵敏度为15 pg/ml真菌DNA。收集从临床分离的84株致病真菌菌株对基因芯片进行试用,结果显示基因芯片的鉴定结果与常规鉴定方法的鉴定结果一致。结论这项技术的建立可以稳定、特异性地实现临床常见致病真菌的高通量鉴定,为进一步检测研究奠定了基础。     

MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues

MicroRNAs (MIRs) are a novel group of conserved short approximately 22 nucleotide-long RNAs with important roles in regulating gene expression. We have established a MIR-specific oligonucleotide microarray system that enables efficient analysis of the expression of the human MIRs identified so far. We show that the 60-mer oligonucleotide probes on the microarrays hybridize with labeled cRNA of MIRs, but not with their precursor hairpin RNAs, derived from amplified, size-fractionated, total RNA of human origin. Signal intensity is related to the location of the MIR sequences within the 60-mer probes, with location at the 5' region giving the highest signals, and at the 3' end, giving the lowest signals. Accordingly, 60-mer probes harboring one MIR copy at the 5' end gave signals of similar intensity to probes containing two or three MIR copies. Mismatch analysis shows that mutations within the MIR sequence significantly reduce or eliminate the signal, suggesting that the observed signals faithfully reflect the abundance of matching MIRs in the labeled cRNA. Expression profiling of 150 MIRs in five human tissues and in HeLa cells revealed a good overall concordance with previously published results, but also with some differences. We present novel data on MIR expression in thymus, testes, and placenta, and have identified MIRs highly enriched in these tissues. Taken together, these results highlight the increased sensitivity of the DNA microarray over other methods for the detection and study of MIRs, and the immense potential in applying such microarrays for the study of MIRs in health and disease.     

A study of nucleotide sequence homology between the nucleic acids of different alphaviruses

Base sequence homologies have been analyzed between the virus-specific nucleic acids of the four different alphaviruses, Semliki Forest (SF) virus, Sindbis (SIN) virus, Chikungunya (CHIK) virus, and O'nyong-nyong (ONN) virus and between different strains of these viruses by RNA-RNA hybridization experiments and by two-dimensional gel electrophoretic analyses of the oligonucleotides generated by RNase T₁ digestion. The following results were obtained: (1) Similar to earlier findings in SIN and SF virus-infected cells two species of single-stranded virus-specific RNA sedimenting at about 42 S and 26 S on sucrose density gradients, respectively, are synthesized in ONN and CHIK virus-infected cells and the 26 S RNA molecules are subgenomic RNA species. (2) Different oligonucleotide patterns were obtained for the genome RNA molecules of the different viruses and none of the oligonucleotides characterized was present in more than one of the viruses. (3) Besides a large number of common oligonucleotides, differences were found in the oligonucleotide patterns of different strains of the same virus, which allowed the identification of the virus strain from the oligonucleotide pattern of its genome RNA. (4) A 13% base sequence homology was detected between CHIK virus and ONN virus by RNA-RNA hybridization, whereas only small sequence homologies of about 1% or less were detected between the other viruses.     

Rapid detection of a plant virus by solution hybridization using oligonucleotide probes

A novel solution hybridization method for the diagnosis of a plant virus was evaluated. Synthetic oligonucleotide probes were used for the detection of potato virus X (PVX) in crude leaf sap extracts by hybridization in solution. Three 40-nucleotide-long oligonucleotide probes complementary to RNA sequences of potato virus X near the 3' end were synthesized. Two probes were 32P-labelled and one biotinylated. The three probes were allowed to form hybrids with the target viral nucleic acid in solution, and the formed hybrids were isolated with the aid of the biotinylated capture probe using avidin polystyrene beads after the reaction. Alternatively, hybrids were captured from the poly(A) tail of the viral RNA on oligo(dT) cellulose. The maximum signal was obtained after 4 h hybridization. About 70% of the maximum signal was obtained after 2 h hybridization. Sensitivity with the two 32P-labelled oligonucleotide probes was 1-5 x 10(7) molecules of PVX RNA. This corresponds to 0.6-3 ng of the virus. Crude leaf sap did not interfere with the detection of the virus. These results suggest that this solution hybridization method permits rapid detection of a plant virus in crude plant sap without sample pretreatment and may thus open new avenues for the development of a nucleic-acid-based ELISA-like diagnostic test for the detection of plant viruses.     

Simultaneous detection and identification of hepatitis A virus and rotavirus by multiplex nucleic acid sequence-based amplification (NASBA) and microtiter plate hybridization system

Human rotavirus and hepatitis A virus (HAV) are two of the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for their detection in foodstuffs. In this study, a multiplex nucleic acid sequence-based amplification (NASBA) system was developed to detect specifically and simultaneously human rotavirus and HAV. Two sets of primers selected from published nucleic acid sequences were used in the NASBA mixture to amplify viral RNA from both viruses. Denaturing gel electrophoresis revealed two distinct RNA products with 268 and 474 nucleotides amplified from rotavirus and HAV, respectively. The specificity of the multiplex NASBA was confirmed by a microtiter plate hybridization and detection system and by Northern blot analysis using specific oligonucleotide probes. The presence of non-homologous nucleic acid and non-target microorganisms did not have any effect on the specificity of the multiplex NASBA. Using the optimized NASBA and microtiter plate hybridization conditions, as little as 400 PFU ml x (-1) of HAV and 40 PFU ml x (-1) of rotavirus were detected. The multiplex NASBA system offers advantages over monoplex virus detection systems in terms of turnaround time and cost-effectiveness.     

高性价比寡核苷酸基因表达谱芯片的制备方法

目的探讨探针长度对寡核苷酸(Oligo)基因芯片杂交信号的影响。方法以大肠杆菌基因表达信息作为实验模型,根据已有大肠杆菌全基因组芯片数据选择覆盖高、中、低杂交信号强度的20个大肠杆菌基因,针对该20个基因分别设计59-mer和70-mer长度Oligo探针,并将2种探针点制在同一张芯片中,同时设阳性对照探针和阴性对照点样液。提取大肠杆菌RNA,经过反转录、扩增、荧光标记后与芯片进行杂交反应,采用激光共聚焦扫描仪扫描芯片,利用数据分析软件提取探针的杂交信号值并进行显著性分析。结果大肠杆菌28SrRNA和18SrRNA条带清晰,无降解带出现,质量合格。芯片杂交结果显示59-mer和70-mer长度探针的杂交效率和杂交信号差异无统计学意义(P=0.9810),阳性对照探针出现阳性杂交信号,阴性对照点样液未检测到杂交信号,符合质控要求。结论59-mer长度探针可用来制备Oligo基因芯片,这不仅降低了基因芯片制作成本,而且将推动基因芯片技术更为普及的应用。     

Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization

Microarrays of virus-specific oligonucleotides may provide a method of screening samples for the presence or absence of a large variety of viruses simultaneously. Influenza viruses are ideal for evaluating such microarrays because of their genetic and host diversity, and the availability of an extensive sequence database. A collection of 476 influenza virus-specific oligonucleotides was spotted onto glass slides as probes. Viral RNAs were reverse transcribed and amplified by PCR, and the products were labeled with cyanine dyes. The presence of viruses and their identities were determined by hybridization. The fluorescence intensities of oligonucleotide spots were highly reproducible within each slide and satisfactorily proportional between experiments. However, the intensities of probe spots completely complementary to target sequences varied from background to saturation. The variations did not correlate with base composition, nucleotide sequence, or internal secondary structures. Therefore, thresholds for determining whether hybridization to a spot should be judged as positive were assigned individually. Considering only positive spots from probes predicted to be monospecific for influenza virus species, subtype, host source, or gene segment, this method made correct identifications at the species, hemagglutinin subtype, and gene segment levels. Monospecific neuraminidase (NA) subtype probes were insufficiently diverse to allow confident NA subtype assignment. Incorporating positive spots from polyspecific probes into the identification scheme gave similar results. Overall, the results demonstrate the potential of microarray-based oligonucleotide hybridization for multiple virus detection.     

Investigating the sensitivity of a fluorescence-based microarray for the detection of fruit-tree viruses

An oligonucleotide microarray for the detection of some fruit-tree viruses was designed and its theoretical detection limit was assessed using Cy3-labelled oligonucleotides. The real sensitivity of the microarray was compared for different kinds of fluorescently labelled targets: (a) cDNA and PCR amplified targets, (b) PCR amplified targets labelled using three different labelling methods. In the first case (a), the number of viral cDNA molecules was below the assessed detection limit of the microarray and only PCR amplified targets were detected. A second comparison (b), done on 3 selected viruses, included indirect labelling, the direct incorporation of labelled-dUTPs, and the use of Cy3-labelled primer. The targets labelled most intensively were produced by the Cy3-primer labelling (2 of 3 viruses) or by the indirect labelling method (1 of 3 viruses), the weakest signal showed targets labelled directly (all 3 viruses). The use of Cy3-primer labelling involved the simplest preparation and the lowest cost, however occasional weak cross-hybridization appeared. The indirect labelling method was of the highest specificity. The probes hybridizing near the 3-end of the targets showed the lowest intensities of fluorescent signal.     

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5' ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.