Improving signal intensities for genes with low-expression on oligonucleotide microarrays

Background  

DNA microarrays using long oligonucleotide probes are widely used to evaluate gene expression in biological samples. These oligonucleotides are pre-synthesized and sequence-optimized to represent specific genes with minimal cross-hybridization to homologous genes. Probe length and concentration are critical factors for signal sensitivity, particularly when genes with various expression levels are being tested. We evaluated the effects of oligonucleotide probe length and concentration on signal intensity measurements of the expression levels of genes in a target sample.     

Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii

Background

DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes.

Results

Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation.

Conclusion

In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression.     

Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays

Background

Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20–60 base) unique sequence tags, or “barcodes”, associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses.

Methodology/Principal Findings

Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5′-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface.

Conclusions/Significance

These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.     

Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis

Key message

Extended antipodal life-span.

Abstract

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.     

Determinants of sensitivity and specificity in spotted DNA microarrays with unmodified oligonucleotides

DNA microarrays with unmodified oligonucleotides are a cost-effective alternative to cDNA microarrays. This study examined how purity, length, homology and GC content of the oligonucleotide probes influence the sensitivity and specificity of the method using cyanobacterial genes. Oligonucleotide purification by high pressure liquid chromatography was omitted without significant reduction in hybridization sensitivity. For two of three genes tested, a reduction in oligonucleotide length did not reduce hybridization sensitivity, and maximum sensitivity was achieved with probes that were 45 nt long. Oligonucleotide probes with 相似文献   

Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

Background

Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.

Results

We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.

Conclusion

A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.     

AtHSP17.8 overexpression in transgenic lettuce gives rise to dehydration and salt stress resistance phenotypes through modulation of ABA-mediated signaling

Key message

Transgenic Arabidopsis and lettuce plants overexpressing AtHSP17.8 showed ABA-hypersensitive but abiotic stress-resistant phenotypes. ABA treatment caused a dramatic induction of early ABA-responsive genes in AtHSP17.8 -overexpressing transgenic lettuce.

Abstract

Plant small heat shock proteins function as chaperones in protein folding. In addition, they are involved in responses to various abiotic stresses, such as dehydration, heat and high salinity in Arabidopsis. However, it remains elusive how they play a role in the abiotic stress responses at the molecular level. In this study, we provide evidence that Arabidopsis HSP17.8 (AtHSP17.8) positively regulates the abiotic stress responses by modulating abscisic acid (ABA) signaling in Arabidopsis, and also in lettuce, a heterologous plant when ectopically expressed. Overexpression of AtHSP17.8 in both Arabidopsis and lettuce leads to hypersensitivity to ABA and enhanced resistance to dehydration and high salinity stresses. Moreover, early ABA-responsive genes, ABI1, ABI5, NCED3, SNF4 and AREB2, were rapidly induced in AtHSP17.8-overexpressing transgenic Arabidopsis and lettuce. Based on these data, we propose that AtHSP17.8 plays a crucial role in abiotic stress responses by positively modulating ABA-mediated signaling in both Arabidopsis and lettuce. Moreover, our results suggest that stress-tolerant lettuce can be engineered using the genetic and molecular resources of Arabidopsis.     

Application of Equilibrium Models of Solution Hybridization to Microarray Design and Analysis

Background

The probe percent bound value, calculated using multi-state equilibrium models of solution hybridization, is shown to be useful in understanding the hybridization behavior of microarray probes having 50 nucleotides, with and without mismatches. These longer oligonucleotides are in widespread use on microarrays, but there are few controlled studies of their interactions with mismatched targets compared to 25-mer based platforms.

Principal Findings

50-mer oligonucleotides with centrally placed single, double and triple mismatches were spotted on an array. Over a range of target concentrations it was possible to discriminate binding to perfect matches and mismatches, and the type of mismatch could be predicted accurately in the concentration midrange (100 pM to 200 pM) using solution hybridization modeling methods. These results have implications for microarray design, optimization and analysis methods.

Conclusions

Our results highlight the importance of incorporating biophysical factors in both the design and the analysis of microarrays. Use of the probe “percent bound” value predicted by equilibrium models of hybridization is confirmed to be important for predicting and interpreting the behavior of long oligonucleotide arrays, as has been shown for short oligonucleotide arrays.     

Microarray profile of differentially expressed genes in a monkey model of allergic asthma

Background

Inhalation of Ascaris suum antigen by allergic monkeys causes an immediate bronchoconstriction and delayed allergic reaction, including a pulmonary inflammatory infiltrate. To identify genes involved in this process, the gene-expression pattern of allergic monkey lungs was profiled by microarrays. Monkeys were challenged by inhalation of A. suum antigen or given interleukin-4 (IL-4) treatment; lung tissue was collected at 4, 18 or 24 h after antigen challenge or 24 h after IL-4. Each challenged monkey lung was compared to a pool of normal, unchallenged monkey lungs.

Results

Of the approximately 40,000 cDNAs represented on the microarray, expression levels of 169 changed by more than 2.5-fold in at least one of the pairwise probe comparisons; these cDNAs encoded 149 genes, of which two thirds are known genes. The largest number of regulated genes was observed 4 h after challenge. Confirmation of differential expression in the original tissue was obtained for 95% of a set of these genes using real-time PCR. Cluster analysis revealed at least five groups of genes with unique expression patterns. One cluster contained genes for several chemokine mediators including eotaxin, PARC, MCP-1 and MCP-3. Genes involved in tissue remodeling and antioxidant responses were also identified as regulated by antigen and IL-4 or by antigen only.

Conclusion

This study provides a large-scale profile of gene expression in the primate lung following allergen or IL-4 challenge. It shows that microarrays, with real-time PCR, are a powerful tool for identifying and validating differentially expressed genes in a disease model.     

Cross-species hybridisation of pig RNA to human nylon microarrays

Background

The objective of this research was to investigate the reproducibility of cross-species microarray hybridisation. Comparisons between same- and cross-species hybridisations were also made. Nine hybridisations between a single pig skeletal muscle RNA sample and three human cDNA nylon microarrays were completed. Three replicate hybridisations of two different amounts of pig RNA, and of human skeletal muscle RNA were completed on three additional microarrays.

Results

Reproducibility of microarray hybridisations of pig cDNA to human microarrays was high, as determined by Spearman and Pearson correlation coefficients and a Kappa statistic. Variability among replicate hybridisations was similar for human and pig data, indicating the reproducibility of results were not compromised in cross-species hybridisations. The concordance between data generated from hybridisations using pig and human skeletal muscle RNA was high, further supporting the use of human microarrays for the analysis of gene expression in the pig. No systematic effect of stripping and re-using nylon microarrays was found, and variability across microarrays was minimal.

Conclusion

The majority of genes generated highly reproducible data in cross-species microarray hybridisations, although approximately 6% were identified as highly variable. Experimental designs that include at least three replicate hybridisations for each experimental treatment will enable the variability of individual genes to be considered appropriately. The use of cross-species microarray analysis looks promising. However, additional validation is needed to determine the specifiCity of cross-species hybridisations, and the validity of results.     

Application of T-DNA activation tagging to identify glutamate receptor-like genes that enhance drought tolerance in plants

Key message

A high-quality rice activation tagging population has been developed and screened for drought-tolerant lines using various water stress assays. One drought-tolerant line activated two rice glutamate receptor-like genes. Transgenic overexpression of the rice glutamate receptor-like genes conferred drought tolerance to rice and Arabidopsis.

Abstract

Rice (Oryza sativa) is a multi-billion dollar crop grown in more than one hundred countries, as well as a useful functional genetic tool for trait discovery. We have developed a population of more than 200,000 activation-tagged rice lines for use in forward genetic screens to identify genes that improve drought tolerance and other traits that improve yield and agronomic productivity. The population has an expected coverage of more than 90 % of rice genes. About 80 % of the lines have a single T-DNA insertion locus and this molecular feature simplifies gene identification. One of the lines identified in our screens, AH01486, exhibits improved drought tolerance. The AH01486 T-DNA locus is located in a region with two glutamate receptor-like genes. Constitutive overexpression of either glutamate receptor-like gene significantly enhances the drought tolerance of rice and Arabidopsis, thus revealing a novel function of this important gene family in plant biology.     

Distinct gene expression profiles of human type 1 and type 2 T helper cells

Background

The development and activation of CD4⁺ helper T cell (Th) subsets with distinct patterns of unbalanced production of cytokines play an important part in infectious, allergic and autoimmune diseases. Human neonatal cord blood CD4⁺ Th cells can be polarized into type 1 or type 2-like effector cells in vitro by culturing them in the presence of interleukin (IL)-12 or IL-4, respectively. We have exploited this experimental system to identify marker genes that are differentially expressed by polarized Th1 and Th2 cells. An oligonucleotide microarray specifically designed to screen for inflammation-related candidate genes was used and the differential expression was further validated with a quantitative real-time RT-PCR method.

Results

In addition to the previously described marker genes of Th cells, we report subtle changes in the expression of several other genes that represent growth factors, receptors and other signaling molecules in polarized Th1 and Th2 cell subsets. Additionally, we describe a novel set of genes as Th1/Th2 differentiation markers for cells activated by anti-CD3 and anti-CD28 antibodies.

Conclusions

This study demonstrates the power of the targeted use of microarrays in combination with quantitative real-time RT-PCR in identifying and validating new marker genes for gene expression studies.