A database on alternative splice forms on the integrated genetic map service (IGMS)

The IGMS is a comprehensive information system that combines the knowledge from genomic sequence, genetic map and genetic disorders databases. This system is updated weekly and focuses on the analysis of EST data. The IGMS identifies UniGene clusters that are differentially expressed in different types of cancer with respect to different reference tissues. The results can be combined with clinical data to asses the potential relevance of specific genes for patient survival or metastatic spread. The second application maps EST with a specific expression profile. Our third application generates a database of alternative splice forms for nine organisms from EST and mRNA sequence data. The results can be used to find splicing patterns specific for certain tissues or tumour types. Availability: http://www.bioinf.mdc-berlin.de/igms/.     

New experimental and computational approaches to the analysis of gene expression.

Public and private EST (Expressed Sequence Tag) programs provide access to a large number of ESTs from a number of plant species, including Arabidopsis, corn, soybean, rice, wheat. In addition to the homology of each EST to genes in GenBank, information about homology to all other ESTs in the data base can be obtained. To estimate expression levels of genes represented in the DuPont EST data base we count the number of times each gene has been seen in different cDNA libraries, from different tissues, developmental stages or induction conditions. This quantitation of message levels is quite accurate for highly expressed messages and, unlike conventional Northern blots, allows comparison of expression levels between different genes. Lists of most highly expresses genes in different libraries can be compiled. Also, if EST data is available for cDNA libraries derived from different developmental stages, gene expression profiles across development can be assembled. We present an example of such a profile for soybean seed development. Gene expression data obtained from Electronic Northern analysis can be confirmed and extended beyond the realm of highly expressed genes by using high density DNA arrays. The ESTs identified as interesting can be arrayed on nylon or glass and probed with total labeled cDNA first strand from the tissue of interest. Two-color fluorescent labeling allows accurate mRNA ratio measurements. We are currently using the DNA array technology to study chemical induction of gene expression and the biosynthesis of oil, carbohydrate and protein in developing seeds.     

Bayesian Hierarchical Modeling and Selection of Differentially Expressed Genes for the EST Data

Summary Expressed sequence tag (EST) sequencing is a one‐pass sequencing reading of cloned cDNAs derived from a certain tissue. The frequency of unique tags among different unbiased cDNA libraries is used to infer the relative expression level of each tag. In this article, we propose a hierarchical multinomial model with a nonlinear Dirichlet prior for the EST data with multiple libraries and multiple types of tissues. A novel hierarchical prior is developed and the properties of the proposed prior are examined. An efficient Markov chain Monte Carlo algorithm is developed for carrying out the posterior computation. We also propose a new selection criterion for detecting which genes are differentially expressed between two tissue types. Our new method with the new gene selection criterion is demonstrated via several simulations to have low false negative and false positive rates. A real EST data set is used to motivate and illustrate the proposed method.     

Analysis of a simulated microarray dataset: Comparison of methods for data normalisation and detection of differential expression (Open Access publication)

Microarrays allow researchers to measure the expression of thousands of genes in a single experiment. Before statistical comparisons can be made, the data must be assessed for quality and normalisation procedures must be applied, of which many have been proposed. Methods of comparing the normalised data are also abundant, and no clear consensus has yet been reached. The purpose of this paper was to compare those methods used by the EADGENE network on a very noisy simulated data set. With the a priori knowledge of which genes are differentially expressed, it is possible to compare the success of each approach quantitatively. Use of an intensity-dependent normalisation procedure was common, as was correction for multiple testing. Most variety in performance resulted from differing approaches to data quality and the use of different statistical tests. Very few of the methods used any kind of background correction. A number of approaches achieved a success rate of 95% or above, with relatively small numbers of false positives and negatives. Applying stringent spot selection criteria and elimination of data did not improve the false positive rate and greatly increased the false negative rate. However, most approaches performed well, and it is encouraging that widely available techniques can achieve such good results on a very noisy data set.     

DNA sequence-based "bar codes" for tracking the origins of expressed sequence tags from a maize cDNA library constructed using multiple mRNA sources

To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence "bar codes" were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects.     

Estimating and comparing the rates of gene discovery and expressed sequence tag (EST) frequencies in EST surveys

MOTIVATION: Expressed sequence tag (EST) surveys are an efficient way to characterize large numbers of genes from an organism. The rate of gene discovery in an EST survey depends on the degree of redundancy of the cDNA libraries from which sequences are obtained. However, few statistical methods have been developed to assess and compare redundancies of various libraries from preliminary EST surveys. RESULTS: We consider statistics for the comparison of EST libraries based upon the frequencies with which genes occur in subsamples of reads. These measures are useful in determining which one of several libraries is more likely to yield new genes in future reads and what proportion of additional reads one might want to take from the libraries in order to be likely to obtain new genes. One approach is to compare single sample measures that have been successfully used in species estimation problems, such as coverage of a library, defined as the proportion of the library that is represented in the given sample of reads. Another single library measure is an estimate of the expected number of additional genes that will be found in a new sample of reads. We also propose statistics that jointly use data from all the libraries. Analogous formulas for coverage and the expected numbers of new genes are presented. These measures consider coverage in a single library based upon reads from all libraries and similarly, the expected numbers of new genes that will be discovered by taking reads from all libraries with fixed proportions. Together, the statistics presented provide useful comparative measures for the libraries that can be used to guide sampling from each of the libraries to maximize the rate of gene discovery. Finally, we present tests for whether genes are equally represented or expressed in a set of libraries. Binomial and chi2 tests are presented for gene-by-gene comparisons of expression. Overall tests of the equality of proportional representation are presented and multiple comparisons issues are addressed. These methods can be used to evaluate changes in gene expression reflected in the composition of EST libraries prepared from different tissue types or cells exposed to different environmental conditions. AVAILABILITY: Software will be made available at http://www.mathstat.dal.ca/~tsusko     

Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer

The discovery of microRNAs (miRNAs) added an extra level of intricacy to the already complex system regulating gene expression. These single-stranded RNA molecules, 18–25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled much interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling in breast tissue. In this study, the expression of five miRNA genes (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and three small nucleolar RNA genes (RNU19, RNU48 and Z30) was examined across malignant, benign and normal breast tissues to determine the most appropriate normalisation strategy. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue.     

抗大肠癌噬菌体单链抗体的筛选及初步鉴定

 应用 3种方法 (肿瘤细胞膜表面和胞内、裸鼠体内和组织切片 ) ,从全人源化的抗大肠癌噬菌体初级抗体库中筛选肿瘤特异性的噬菌体单链抗体 (Sc Fv) .在肿瘤细胞经过 3轮亲和选择 ,回收结合胞膜和内化进入胞内的噬菌体 ,得到抗肿瘤噬菌体单链抗体的富集倍数为 430倍 ;荷瘤裸鼠体内注入初级抗体库后 ,在不同时刻点处死裸鼠 ,回收肿瘤组织内的噬菌体 ,其回收率在 2 4 h时最高 ;初级抗体库与大肠癌组织切片亲和选择后 ,从冰冻组织切片上比从石蜡组织切片上回收得到的噬菌体高出约 1 .6倍 .从上述方法挑选单克隆 ,经 ELISA筛选抗大肠癌阳性噬菌体克隆株 ,分离得到 5个对大肠癌细胞反应较好的单克隆噬菌体单链抗体 .进一步用细胞 ELISA检测对各种肿瘤细胞的特异性反应 ,其中 4个对大肠癌细胞有很好的特异性 ,1个克隆对所有肿瘤细胞均有反应 .因此 ,3种方法用于筛选抗大肠癌噬菌体初级抗体库是有效的 ,具有推广和应用价值 .     

Variation in tissue-specific gene expression among natural populations

Background

Variation in gene expression is extensive among tissues, individuals, strains, populations and species. The interactions among these sources of variation are relevant for physiological studies such as disease or toxic stress; for example, it is common for pathologies such as cancer, heart failure and metabolic disease to be associated with changes in tissue-specific gene expression or changes in metabolic gene expression. But how conserved these differences are among outbred individuals and among populations has not been well documented. To address this we examined the expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish Fundulus heteroclitus using a highly replicated experimental design.

Results

Half of the genes (48%) were differentially expressed among individuals within a population-tissue group and 76% were differentially expressed among tissues. Differences among tissues reflected well established tissue-specific metabolic requirements, suggesting that these measures of gene expression accurately reflect changes in proteins and their phenotypic effects. Remarkably, only a small subset (31%) of tissue-specific differences was consistent in all three populations.

Conclusions

These data indicate that many tissue-specific differences in gene expression are unique to one population and thus are unlikely to contribute to fundamental differences between tissue types. We suggest that those subsets of treatment-specific gene expression patterns that are conserved between taxa are most likely to be functionally related to the physiological state in question.     

Expressed sequence tag analysis of expression profiles of zebrafish testis and ovary

In the present study, two gonad cDNA libraries from zebrafish testes and ovaries were constructed and a total of 1025 expressed sequence tag (EST) clones were generated from the two libraries: 501 from the testis library and 524 from the ovary library. A total of 641 of the EST clones were identified to share significant sequence identity with known sequences in GenBank, representing at least 478 different zebrafish genes. In order to understand the molecular compositions of the two gonad organs, the expression profiles of the identified clones in these two gonad cDNA libraries were analyzed. Both gonad libraries have a higher portion of clones for nuclear proteins and a lower portion for proteins in translational machinery, cytoskeleton and mitochondria than our previously characterized whole-adult cDNA library. Most abundant cDNA clones in the two gonad libraries were identified and over 10% of ovary clones were found to encode egg membrane proteins (zona pellucida or ZP proteins). Furthermore, the testis library showed a more even distribution of cDNA clones with relatively fewer abundant clones that tend to contribute redundant clones in EST projects; thus, the testis library can supply more unique and novel cDNA sequences in a zebrafish EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the gonad development in zebrafish. Eleven potential clones were selected to analyze their expression patterns by Northern blot hybridization. Most of them showed a specific or predominant expression in the expected testis or ovary tissue. At last, four of the clones were found, by section in situ hybridization, to be expressed specifically in the germ cells of the testis or ovary and thus they are suitable molecular markers for analyses of spermatogenesis and oogenesis.     

Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues.

A four-step procedure for the efficient and systematic mining of whole EST libraries for differentially expressed genes is presented. After eliminating redundant entries from the EST library under investigation (step 1), contigs of maximal length are built upon each remaining EST using about 4 000 000 public and proprietary ESTs (step 2). These putative genes are compared against a database comprising ESTs from 16 different tissues (both normal and tumour affected) to determine whether or not they are differentially expressed (step 3; electronic northern). Fisher's exact test is used to assess the significance of differential expression. In step 4, an attempt is made to characterise the contigs obtained in the assembly through database comparison. A case study of the CGAP library NCI_CGAP_Br1.1, a library made from three (well, moderately, and poorly differentiated) invasive ductal breast tumours (2126 ESTs in total) was carried out. Of the maximal contigs, 139 were found to be significantly (alpha = 0.05) over-expressed in breast tumour tissue, while 13 appeared to be down-regulated.