表达谱基因芯片的可靠性验证分析

cDNA芯片是一项新兴的能评估检测全范围mRNA表达水平变化的技术。通过同种组织RNA自身比较实验及不同组织RNA的差异分析实验对cDNA芯片实验的重复性进行检验,利用相关系数(correlation coefficient,R)、变异系数(coefficient of variation,CV)和假阳性率(false positiver ate,FPR)分析eDNA芯片数据的可靠程度,对cDNA芯片实验数据作了整体的评估。结果证实,该芯片系统得到的cDNA表达谱数据相关系数一般大于0．9,平均变异系数15％左右,假阳性率控制在3％以内。还提出一致率(consistence rate,CR)的概念,作为衡量cDNA芯片系统重复性的新参数,同时阐述了该参数优于目前常用的相关系数及变异系数的特点。另外,通过比较芯片制备中点样浓度、mRNA和总RNA以及不同批次芯片和不同标记过程对实验的影响,来分析芯片数据的系统误差来源。并提出重复两次实验,可以克服绝大部分实验系统引入的假阳性。     

The influence of RNA integrity,purity and cDNA labelling on glass slide cDNA microarray image quality

The accuracy of gene expression measurements generated using cDNA microarrays is dependent on the quality of the image generated following hybridization of fluorescently labelled cDNA. It is not known how this image is influenced by sample preparation factors which such as RNA quality, cDNA synthesis and labelling efficiency. In this study we used a simple metric based on the ratio of the total feature (F) and background (B) fluorescence, which correlates with the visual assessment of 60 microarray images, to determine the influence of sample preparation on image quality. Results indicate that RNA purity (A260/A280) and integrity (18S:28S ratio) do not strongly influence microarray image quality. cDNA having an nucleotide to dye ratio greater than 100 produced poor microarray images, however, cDNA labelled more efficiently was not a guarantee of a better image. The data also indicate that the array image quality is not improved by loading more cDNA into the hybridization mixture however poor image quality did result from a disproportionate amounts of Cy5 and Cy3 labelled cDNA. This study provides insight into the source of variation in microarray image analysis introduced during sample preparation and will assist in the standardisation of cDNA glass slide microarray protocols.     

Sensitivity of 70-mer oligonucleotides and cDNAs for microarray analysis of gene expression in Arabidopsis and its related species

Synthetic oligonucleotides (oligos) represent an attractive alternative to cDNA amplicons for spotted microarray analysis in a number of model organisms, including Arabidopsis, C. elegans, Drosophila, human, mouse and yeast. However, little is known about the relative effectiveness of 60-70-mer oligos and cDNAs for detecting gene expression changes. Using 192 pairs of Arabidopsis thaliana cDNAs and corresponding 70-mer oligos, we performed three sets of dye-swap experiments and used analysis of variance (anova) to compare sources of variation and sensitivities for detecting gene expression changes in A. thaliana, A. arenosa and Brassica oleracea. Our major findings were: (1) variation among different RNA preparations from the same tissue was small, but large variation among dye-labellings and slides indicates the need to replicate these factors; (2) sources of variation were similar for experiments with all three species, suggesting these feature types are effective for analysing gene expression in related species; (3) oligo and cDNA features had similar sensitivities for detecting expression changes and they identified a common subset of significant genes, but results from quantitative RT-PCR did not support the use of one over the other. These findings indicate that spotted oligos are at least as effective as cDNAs for microarray analyses of gene expression. We are using oligos designed from approximately 26,000 annotated genes of A. thaliana to study gene expression changes in Arabidopsis and Brassica polyploids.     

一种用于基因芯片可靠的双轮RNA线性扩增技术

DNA微阵列能在一次实验中检测成千上万个基因的表达情况, 有助于阐明疾病发生的分子机制及发现新的诊治靶标.但常规方法需要大量RNA, 因而基于T7 RNA线性扩增技术逐渐成为微阵列表达谱实验中最常用的探针制备方法.本方法将实验步骤进一步改进,增加额外的一轮体外转录,并结合Klenow酶标记技术来制备cDNA靶标和寡核苷酸芯片杂交.从纳克量级的总RNA起始,本方法和常规的RNA单轮线性扩增法相比,仍然准确地保留了约70%的基因表达信息.同一RNA样本的自身比较实验及重复实验结果也显示,该方法具有较高的可靠性和重复性.RNA双轮体外扩增法需要的起始RNA相对于常规的单轮扩增法减少了很多（10 ng甚至更少）,因而非常适合分析那些只能提供微量RNA的样本.     

Production of bulk amounts of universal RNA for DNA microarrays

In DNA microarray technology, repeatability and reliability are very important to compare multiple RNA samplesfrom different experiments. The application of common or universal RNA as a standard control equalizes the differences in hybridization parameters and array variations. For this purpose, high-quality reference RNA is necessary in bulk amounts. A novel approach was developed to get milligrams of sense or antisense RNA, starting from micrograms of pooled total RNA from different cell lines, tissues, or organisms. This method is inexpensive and allows further labeling procedures using poly(dT) or random oligomers as primers. In addition, amplified, sense reference RNA is suitable for standard labeling protocols, while the antisense reference RNA can be used with antisense RNA from the linear sample amplification method. Here we produced universal RNA for human, rat, and alfalfa and demonstrated the quality using specific cDNA microarrays.     

Linear amplification of catalyzed reporter deposition technology on nylon membrane microarray

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal qualityfrom the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 microg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 microg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.     

Differences in gene expression between B-cell chronic lymphocytic leukemia and normal B cells: a meta-analysis of three microarray studies

MOTIVATION: A major focus of current cancer research is to identify genes that can be used as markers for prognosis and diagnosis, and as targets for therapy. Microarray technology has been applied extensively for this purpose, even though it has been reported that the agreement between microarray platforms is poor. A critical question is: how can we best combine the measurements of matched genes across microarray platforms to develop diagnostic and prognostic tools related to the underlying biology? RESULTS: We introduce a statistical approach within a Bayesian framework to combine the microarray data on matched genes from three investigations of gene expression profiling of B-cell chronic lymphocytic leukemia (CLL) and normal B cells (NBC) using three different microarray platforms, oligonucleotide arrays, cDNA arrays printed on glass slides and cDNA arrays printed on nylon membranes. Using this approach, we identified a number of genes that were consistently differentially expressed between CLL and NBC samples.     

Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation

There are many sources of systematic variation in cDNA microarray experiments which affect the measured gene expression levels (e.g. differences in labeling efficiency between the two fluorescent dyes). The term normalization refers to the process of removing such variation. A constant adjustment is often used to force the distribution of the intensity log ratios to have a median of zero for each slide. However, such global normalization approaches are not adequate in situations where dye biases can depend on spot overall intensity and/or spatial location within the array. This article proposes normalization methods that are based on robust local regression and account for intensity and spatial dependence in dye biases for different types of cDNA microarray experiments. The selection of appropriate controls for normalization is discussed and a novel set of controls (microarray sample pool, MSP) is introduced to aid in intensity-dependent normalization. Lastly, to allow for comparisons of expression levels across slides, a robust method based on maximum likelihood estimation is proposed to adjust for scale differences among slides.     

猪瘟病毒基因表达谱芯片的可靠性研究

通过同种组织RNA自身比较实验及不同组织RNA的差异分析实验对猪瘟病毒(classic swine fever virus, CSFV)cDNA芯片实验的重复性进行检验.利用相关系数(correlationcoefficient, R)、变异系数(coefficientofvariation, CV)和假阳性率(falsepositiverate, FPR)分析猪瘟病毒cDNA芯片数据的可靠程度,对cDNA芯片实验数据作了整体的评估.结果证实,该芯片系统得到的猪瘟病毒cDNA表达谱数据相关系数一般大于0.9,假阳性率控制在2 %以内.另外,通过比较猪瘟病毒芯片制备中点样浓度、mRNA和总RNA以及不同标记过程对实验的影响,来分析猪瘟病毒芯片数据的系统误差来源,得出结果:重复两次实验,可以克服绝大部分实验系统引入的假阳性.     

Impact of RNA extraction from limited samples on microarray results

To move microarray technology into the diagnostic realm, the impact of technical parameters, such as sample preparation and RNA extraction, needs to be understood and minimized. We evaluated the impact of two RNA extraction methods, DNase treatment and the amount of hybridized cDNA probe, on the outcome of microarray results. The results for both RNA extraction methods were comparable, although one method resulted in residual DNA that slightly affected the microarray results. As little as one microgram of total RNA could be used to synthesize a cDNA probe and resulted in a gene expression profile that was similar to one produced using 5 micrograms total RNA, even though the overall signal intensity was lower. These experiments illustrate that microarray technology holds great promise for the use of limited clinical samples in the diagnostic setting.     

Technical evaluation of cDNA microarrays

The aims were to evaluate the common reference design approach in microarray experiments and to evaluate the technical performance and the normalisation of cDNA microarrays with a limited number of spots. Total RNA from 3 normal and 3 tumour sample biopsies were used for synthesis of amino-allyl labelled cRNA. Equal amounts of cRNA from all samples were mixed as reference. After conjugation of cRNA with fluorophores (Cy3/Cy5), each individual tumour cRNA was hybridised to a cDNA microarray together with reference cRNA, scanned and analysed. We show that our procedures for producing cDNA microarrays are reproducible. The concordance between duplicated spots and replicate hybridisation was found to be high. We have demonstrated that our cDNA microarrays are of a high technical quality. The majority of the cDNA microarrays had low local spot background levels. Despite the high background levels for some local spots, variation could be minimized by locally weighted scatter plot smooth normalisation (LOWESS), which we showed was also suitable for normalisation of cDNA microarrays with a limited number of probes.