Effective dimension reduction methods for tumor classification using gene expression data

MOTIVATION: One particular application of microarray data, is to uncover the molecular variation among cancers. One feature of microarray studies is the fact that the number n of samples collected is relatively small compared to the number p of genes per sample which are usually in the thousands. In statistical terms this very large number of predictors compared to a small number of samples or observations makes the classification problem difficult. An efficient way to solve this problem is by using dimension reduction statistical techniques in conjunction with nonparametric discriminant procedures. RESULTS: We view the classification problem as a regression problem with few observations and many predictor variables. We use an adaptive dimension reduction method for generalized semi-parametric regression models that allows us to solve the 'curse of dimensionality problem' arising in the context of expression data. The predictive performance of the resulting classification rule is illustrated on two well know data sets in the microarray literature: the leukemia data that is known to contain classes that are easy 'separable' and the colon data set.     

Differential gene expression of rice roots inoculated with the diazotroph Herbaspirillum seropedicae

Background and aims

Rice (Oryza sativa L.) is the primary source of carbohydrate for the majority of the World's population. Herbaspirillum seropedicae is a diazotroph that lives within and on the surface of rice roots. It can promote the growth of rice, partly by supplying it with fixed nitrogen.

Methods

To better understand the rice–H. seropedicae interaction, cDNA libraries from rice roots either inoculated (RRCH) or uninoculated (RRSH) with the diazotroph were obtained and analysed.

Results

Potential differentially expressed genes identified from the libraries encoded a metallothionein-like protein type 1, a NOD26-like membrane integral protein ZmNIP2-1, a thionin family protein, an oryzain gamma chain precursor, stress-associated protein 1 (OsISAP1), probenazole-inducible protein PBZ1 and auxin- and ethylene-responsive genes. Differential expression was analysed by qRT-PCR for some of these genes and confirmed in most cases. The expression of stress- and defence-related genes coding for thionins, PBZ1 and OsISAP1 was repressed, while expression of a metallothionein gene was induced by inoculation with H. seropedicae. In contrast, expression of auxin-responsive genes was repressed, while expression of ethylene genes was either repressed or induced. The possible involvement of these and other genes in plant-bacterial interactions is discussed.

Conclusions

The decrease in expression of the defence-related proteins PBZ1 and thionins in the rice–H. seropedicae association, suggests that the bacteria modulate plant defence responses during colonisation. The expression of genes responsive to auxin and ethylene also appears to be regulated by the bacteria.     

Nitrogen availability affects hydraulic conductivity of rice roots,possibly through changes in aquaporin gene expression

Background and aims

Nitrogen (N) availability affects water uptake from the roots, which decreases upon N deprivation and increases upon resupply. The aim of this study was to reveal possible mechanisms of regulation of water transport in roots through physiological and morphological adaptations to N availability.

Methods

The effects of continuous N deprivation and following resupply on root morphology, osmotic hydraulic conductivity, and expression of genes for aquaporins (water channels) were examined in rice (Oryza sativa L.) plants. The effect of local N availability was examined by using a split-root system.

Results

N deprivation decreased the expression of root-specific aquaporin genes, whereas N resupply increased their expression. Changes in aquaporin gene expression were correlated with changes in hydraulic conductivity. N deprivation increased dry matter allocation to the roots. In a split-root experiment, the expression of root-specific aquaporin genes was down-regulated in the N-deprived half, whereas it was up-regulated in the N-supplied half.

Conclusion

Our results suggest that expression of genes for root-specific aquaporins underlies the changes in conductivity during continuous N deprivation and resupply. Rice plants seem to adapt to N availability through coordinated adjustment of root proliferation and abundance of aquaporins.     

Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data

A large number of computational methods have been developed for analyzing differential gene expression in RNA-seq data. We describe a comprehensive evaluation of common methods using the SEQC benchmark dataset and ENCODE data. We consider a number of key features, including normalization, accuracy of differential expression detection and differential expression analysis when one condition has no detectable expression. We find significant differences among the methods, but note that array-based methods adapted to RNA-seq data perform comparably to methods designed for RNA-seq. Our results demonstrate that increasing the number of replicate samples significantly improves detection power over increased sequencing depth.     

Proteomic changes in rice leaves during development of field-grown rice plants

Of the numerous factors affecting rice yield, how solar radiation is transformed into biomass through rice leaves is the most important. We have analyzed proteomic changes in rice leaves collected from six different developing stages (vegetative to ripening). We studied protein expression profiles of rice leaves by running two-dimensional gel electrophoresis. Differential protein expression among the six phases were analyzed by image analysis, which allowed the identification of 49 significantly different gel spots. The spots were further verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, in which 89.8% of them were confirmed to be rice proteins. Finally, we confirmed some of the interesting rice proteins by immunoblotting. Three major conclusions can be drawn from these experimental results. (i) Protein expression in rice leaves, at least for high or middle abundance proteins, is attenuated during growth (especially some chloroplast proteins). However, the change is slow and the expression profiles are relatively stable during rice development. (ii) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major protein in rice leaves, is expressed at constant levels at different growth stages. Interestingly, a high ratio of degradation of the RuBisCO large subunit was found in all samples. This was confirmed by two approaches, mass spectrometry and immunoblotting. The degraded fragments are similar to other digested products of RuBisCO mediated by free radials. (iii) The expression of antioxidant proteins such as superoxide dismutase and peroxidase decline at the early ripening stage.     

Statistical analysis of global gene expression data: some practical considerations

Applying appropriate error models and conservative estimates to microarray data helps to reduce the number of false predictions and allows one to focus on biologically relevant observations. Several key conclusions have been drawn from the statistical analysis of global gene expression data: it is worth keeping core information for each experiment, including raw and processed data; biological and technical replicates are needed; careful experimental design makes the analysis simpler and more powerful; the choice of the similarity measure is nontrivial and depends on the goal of an experiment; array information must be complemented with other data; and gene expression studies are 'hypothesis generators'.     

The application of DNA microarrays in gene expression analysis

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.     

Global gene expression analysis of a rice high-tillering dwarf mutant

Plant height and tiller number are indispensible for the establishment of grain production in rice (Oryza sativa L.). A new rice mutant high-tillering dwarf 3 (htd3) exhibiting more tiller number and shorter culm length than the wild-type Guichao 2 (GC2, an indica cultivar) was used to investigate the global gene expression patterns at days after germination 25 (DAG25) and DAG60. In this study, we identified 305 and 987 genes with at least twofold change in gene expression level at DAG25 and DAG60 respectively using the rice microarray chip. Gene ontology enrichment analysis of these twofold change regulated genes revealed that large numbers of genes were involved in binding activity, catalytic activity and metabolic process. The chip results also showed that some of the regulated genes involved in diverse molecular pathways, including gibberellin pathway, brassinosteroid pathway and auxin signal, had significant differences in gene expression abundance at DAG60. This genome-wide gene expression analysis could provide a new opportunity to uncover the regulation mechanisms of the development of culm and tiller, two important components of yields in rice.     

Statistical methods for the practical clinical application of morphometric measurements

Four statistical methods are presented to determine the practical clinical value of measurements made from malignant tumors and to translate these measurements into a prediction of survival for each patient: the Cox statistical model, which must be derived from a data base of cases with known outcome; the null-rank test, a modified rank-sum test that provides an overall measure of the effectiveness of the Cox model; the predicted survival curve, an estimate of survival derived for each new patient from measurements of the primary tumor; and the standard error of measurement, an empirical method for estimating the variability introduced into predicted survival by errors in measurement. The value of these statistical methods was demonstrated by application to 200 cases of human intraocular melanoma, with the two predictive morphometric measurements used being the standard deviation of nucleolar area (SDNA) and the largest tumor dimension (LTD) derived from a single histologic slide of each tumor. Sufficient references and mathematical details are provided to allow anyone with moderate skills as a computer programmer to construct or obtain all of the relevant algorithms.     

Aluminum triggers broad changes in microRNA expression in rice roots

MicroRNAs are small 21-nucleotide RNA molecules with regulatory roles in development and in response to stress. Expression of some plant miRNAs has been specifically associated with responses to abiotic stresses caused by cold, light, iron, and copper ions. In acid soils, aluminum solubility increases, thereby causing severe damage to plants. Although physiological aspects of aluminum toxicity in plants have been well characterized, the molecular mediators are not fully elucidated. There have been no reports about miRNA responses to aluminum stress. Modulation of miRNA expression may constitute a key element to explain the mechanisms implicated in aluminum toxicity and tolerance. We examined the expression of at least one miRNA member from each miRNA family in rice roots of Oryza sativa spp indica cv. Embrapa Taim and Oryza sativa spp japonica cv. Nipponbare under high concentrations of aluminum. Forty-six miRNA families were effectively detected by quantitative PCR. Among these, 13 were down-regulated and six were up-regulated in roots of the Nipponbare cultivar after 8 h of aluminum treatment. In roots of the Embrapa Taim cultivar, five miRNAs were down-regulated and three were up-regulated. Analyses of their putative targets suggest that these rice miRNAs are involved in the regulation of various metabolic pathways in response to high concentrations of aluminum.     

Dot-blot-SNP analysis for practical plant breeding and cultivar identification in rice

We report dot-blot hybridization with allele-specific oligonucleotides for single nucleotide polymorphisms (SNPs) analysis to be applicable for practical plant breeding and cultivar identification. Competitive hybridization of a digoxigenin-labeled oligonucleotide having the sequence of a mutant allele (or a wild-type allele) together with an unlabeled oligonucleotide having the sequence of a wild-type allele (or a mutant allele) was highly effective to reduce background signals in dot-blot hybridization. All 100 tested genes (200 alleles) in rice having SNPs or insertions/deletions were detected in an allele-specific manner. Genotypes of 43 rice cultivars were identified by this technique, and eight SNP markers were found to be sufficient for distinguishing all the cultivars from each other. Dot-blot analysis was also applied to genotyping of Wx and Sd1 of F₄ plants in a conventional breeding program. Since dot-blot analysis with competitive hybridization provides a highly reliable, simple, and cost-effective technique for SNP analysis of a large number of samples, this technique is expected to realize the practical use of a novel breeding method, in which plants or breeding lines are selected by SNP analyses of many genes in a laboratory.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The microarray analysis for gene expression in haploids and diploids derived from twin-seedling rice

In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Differ- ent degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 se- quences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the bio- logical process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.     

Sequence analysis of rice rubi3 promoter gene expression cassettes for improved transgene expression

In a construct containing a GUS reporter gene driven by the 5′ regulatory elements from rubi3, expression was enhanced 4-fold when a 20-nucleotide (nt) GUS 5′ untranslated sequence was replaced with 9 nt sequences derived from rubi3′s second exon. The roles of the sequences immediately upstream from the GUS translation initiation codon, and their significance in gene expression, were investigated. Sequence analysis suggests that complementarity between sequences immediately 5′ of a translation initiation codon and the rice 17S rRNA may be responsible for the reduction in protein levels from constructs containing the GUS leader sequence. The results demonstrate an affect sequences immediately upstream from transgenic coding sequences have on expression, and when using the rubi3 5′ regulatory sequence in particular.     

The microarray analysis for gene expression in haploids and diploids derived from twin-seedling rice

In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Different degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 sequences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the biological process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.