一种标记cDNA芯片探针的新方法

探讨mRNA长片段反转录PCR技术(RT-LDPCR)在cDNA芯片微量探针标记和信号放大中的应用.首先提取BEP2D细胞的总RNA,然后用两种不同的方法进行标记,一种为RT-LDPCR,用荧光素Cy3-dCTP进行标记;另一种为传统的RNA反转录,用荧光素Cy5-dCTP进行标记.将两种方法标记好的探针等量混合后与含有440个点(44个基因)的cDNA芯片同时杂交,发现二者具有很高的一致性(0.5＜Cy3/Cy5＞2.0).由于RNA反转录法为cDNA芯片探针标记的传统方法,从而验证了RT-LDPCR用于cDNA芯片探针标记的可行性.RT-LDPCR具有对样品总RNA的需要量少和可对样品中信号进行放大的优点,特别适合于对材料来源受到限制的RNA进行标记.     

Developmental aberrations of liver gene expression in bovine fetuses derived from somatic cell nuclear transplantation

Cloning by somatic cell nuclear transfer (NT) has been accomplished. However, the process itself is inefficient since most clones die before birth and survivors often display various anomalies. In an effort to determine global expression profiles of developmentally regulated liver genes in NT bovine fetuses, we employed a custom-made bovine liver complementary DNA (cDNA) microarray. The NT fetuses in early pregnancy were derived from cumulus cells as the nuclear donor cells. Normal fetuses were derived from in vitro fertilization (IVF) and artificial insemination (AI). Gene expression levels in NT, IVF, and AI fetal livers were obtained by comparing individual fetal liver samples with that of adult liver of nonpregnant cycling cows. Statistical analyses of the expression data showed widespread dysregulation of developmentally important genes in the three NT fetuses examined. It was found that the number of dysregulated genes was within a range of 3.5-7.7% of the tested genes in the NT fetal livers. The analyses revealed that one NT fetus was markedly different in liver gene expression profile from the other two NT fetal livers in which the expression profiles were highly correlated. Thus, our findings demonstrate that widespread dysregulation of liver genes occurs in the developing liver of NT bovine fetuses. It is possible that inappropriate genomic reprogramming after NT is a key factor associated with abnormal gene expressions in the livers of NT fetuses, whereas distinct expression patterns between the fellow cloned fetuses likely have resulted from variable epigenetic status of the donor nuclei.     

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.     

Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation,using expressed sequence tags (ESTs) analysis and cDNA microarrays

To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified.The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.     

Statistical design of reverse dye microarrays

MOTIVATION: In cDNA microarray experiments all samples are labelled with either Cy3 dye or Cy5 dye. Certain genes exhibit dye bias-a tendency to bind more efficiently to one of the dyes. The common reference design avoids the problem of dye bias by running all arrays 'forward', so that the samples being compared are always labelled with the same dye. But comparison of samples labelled with different dyes is sometimes of interest. In these situations, it is necessary to run some arrays 'reverse'-with the dye labelling reversed-in order to correct for the dye bias. The design of these experiments will impact one's ability to identify genes that are differentially expressed in different tissues or conditions. We address the design issue of how many specimens are needed, how many forward and reverse labelled arrays to perform, and how to optimally assign Cy3 and Cy5 labels to the specimens. RESULTS: We consider three types of experiments for which some reverse labelling is needed: paired samples, samples from two predefined groups, and reference design data when comparison with the reference is of interest. We present simple probability models for the data, derive optimal estimators for relative gene expression, and compare the efficiency of the estimators for a range of designs. In each case, we present the optimal design and sample size formulas. We show that reverse labelling of individual arrays is generally not required.     

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.     

Characterization of gene expression profiles in early bovine pregnancy using a custom cDNA microarray

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placental-specific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.     

基因芯片技术筛选家蝇抗菌肽相关基因

用生物学软件对GenBank中部分昆虫抗菌肽基因编码区保守域设计探针, 用直接点样法将探针点印在特制玻片上构建寡核苷酸(Oligonucleotide, oligo)探针微阵列; 提取诱导后24 h的家蝇三龄幼虫脂肪体总RNA, 逆转录成cDNA并标记上荧光标记物Cy3, 与构建的oligo探针微阵列杂交, 经洗片、扫描处理后进行数据分析。结果在两次重复实验中均检测到有效杂交信号的基因点有15个(不包括阳性对照基因), 为进一步发现其新基因提供了依据。     

Identification of development and tissue-specific gene expression in the fathead minnow Pimephales promelas, Rafinesque using computational and DNA microarray methods

The fathead minnow Pimephales promelas serves as a model organism for assessing the effects of environmental contaminants on early life stage growth and development. Yet, the utilization of genomic tools has been hindered by the lack of genome sequence and genomic information known from this model species. Utilizing published cDNA library sequences, the authors used sequence similarity to compare 4105 cDNAs isolated from fathead minnow fry (<14 days old) with over 250 000 adult cDNA sequences derived from whole body and various tissue types. The objectives of the computational subtraction were to (1) assess the extent of sequence similarity between developing and adult cDNA libraries and (2) predict which cDNA clones are expressed only in developing organisms. The results of the computational predictions were assessed through the construction of a development‐specific DNA microarray targeting all 4105 sequences in the fry cDNA library as well as 56 known mRNAs in P. promelas. Gene expression was determined by comparing total RNA isolated from fry with total RNA isolated from adult samples (whole animal, kidney, liver, brain, ovary and testes). The results showed that 1381 of the targeted fry cDNA sequences (34%) displayed expression across all sample comparisons, and of these, only 166 genes were found to harbour fry‐specific expression (i.e. no expression in adult samples). Of note, 69% of the genes computationally predicted to be fry specific were found across all experimental results; yet, only 27% of the computationally predicted fry‐specific sequences were experimentally confirmed to be fry specific. An important result was the identification of many novel mRNA sequences specific to the developing minnow, which lack homology with any other known sequence. In addition, the study results included tissue‐specific expression in adult samples. These results demonstrate the capabilities and limitations of inter‐library sequence comparisons as a predictor of gene activity in non‐sequenced organisms and tissues, as well as DNA microarray gene expression studies in non‐sequenced organisms.     

利用表达谱基因芯片筛选溃疡性结肠炎差异表达基因

目的:利用人类全基因组表达谱芯片技术,分析溃疡性结肠炎患者和健康者基因表达谱差异,筛选出溃疡性结肠炎相关基因。方法:采用Trizol法提取8例溃疡性结肠炎患者和8例健康对照者结肠粘膜组织总RNA并纯化,逆转录合成c DNA,利用荧光染料Cy3标记aa UTP,转录合成标记的c RNA,并与Agilent人类全基因组表达谱芯片杂交,扫描荧光信号图像,对芯片原始数据进行归一化处理,利用倍数差异和t检验计算筛选出相关差异表达基因,采用DAVID在线分析系统进行基因的功能注释和关联分析,明确差异基因的生物学功能,并对部分差异表达基因进行实时荧光定量PCR验证。结果:筛查出溃疡性结肠炎结肠粘膜组织差异表达基因4132个,其中上调基因2004个,下调基因2128个。选取6条差异表达基因进行PCR验证,结果有3条基因表达上调,3条基因表达下调,表达趋势与芯片结果一致。结论:溃疡性结肠炎患者与健康对照者基因表达存在明显差异,分析这些差异表达基因有助于我们探索溃疡性结肠炎的发病机制,为疾病的治疗提供理论依据。     

用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因

以基因表达谱芯片对人正常肝及肝癌组织基因表达的差异性进行了研究比较。奖４０９６条人ｃＤＮＡ用点样仪点在特制玻片上制备成表达谱芯片;利用肝和肝癌组织的ｍＲＮＡ通过逆转录方法,将Ｃｙ３和Ｃｙ５２种荧光分别标记到两种组织的ｃＤＮＡ上,制备成ｃＤＮＡ探针,并与表达谱芯片进行杂交及扫描,重复４次实验,通过计算机数据处理判定基因是否在上述２种组织中有表达差异,筛选出差异表达的基因共９０３条。基因芯片技术可同时     

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNA later at 4 degrees C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.     

Expression of lipoprotein lipase associated with lung adenocarcinoma tissues

Lipoprotein lipase (LPL) plays a key role in the lipid metabolism and transporting. It can catalyze the hydrolysis of chylomicron and very low-density lipoprotein triglyceride. Moreover, the abnormality of LPL associates with many pathophysiological conditions. Herein cDNA microarray and Northern blots analysis were used to study the expression of lipoprotein lipase in lung adenocarcinoma tissues. There were 113 genes of all tested blots in cDNA microarray expressed lowly. LPL gene is expressed lowly at the average ratio 0.26 (Cy5/Cy3) in lung adenocarcinoma tissues over controls. Northern blots confirmed those changes detected from the cDNA microarray and suggested that low expression of LPL may play an important role in the lung adenocarcinoma development.