Nonsense-mediated mRNA decay in barley mutants allows the cloning of mutated genes by a microarray approach.

We have previously described a microarray approach to identify and clone genes from mutants of higher organisms. In the method cDNA of two mutants with similar phenotype are competitively hybridized to DNA clones arrayed on a glass slide. Clones corresponding to an mRNA that is not expressed in one of the strains due to a mutation will be specifically highlighted in the hybridization, which provides a possibility to identify and eventually clone the mutated gene. The approach is dependent on mutations that affect the amount of mRNA. Nonsense mutations, which prematurely terminate translation, can be such mutations as a surveillance system known as nonsense-mediated decay (NMD) has been developed by organisms to reduce the abundance of mRNA with nonsense codons. In the present study, we have analysed the barley (Hordeum vulgare L.) magnesium chelatase mutants xantha-f26, xantha-f27 and xantha-f40 in order to investigate the presence of NMD in barley, as well as the importance of the position of the stop codon for NMD. Both nonsense-mutants xantha-f27 and xantha-f40, but not the missense mutant xantha-f26, showed NMD. This was not expected for xantha-f27 as its mutation is in the last exon of the gene. We conclude the NMD expands the number of mutants that can be used for gene cloning by our described microarray approach.     

Characterization of eight barley xantha-f mutants deficient in magnesium chelatase.

Magnesium chelatase (EC 6.6.1.1) catalyses the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway. The enzyme is composed of three different subunits of approximately 40, 70 and 140 kDa. In barley (Hordeum vulgare L.) the subunits are encoded by the genes Xantha-h, Xantha-g and Xantha-f. In the 1950s, eight induced xantha-f mutants were isolated. In this work we characterized these mutations at the DNA level and provided explanations for their phenotypes. The xantha-f10 mutation is a 3 bp deletion, resulting in a polypeptide lacking the glutamate residue at position 424. The leaky mutation xantha-f26 has a missense mutation leading to a M632R exchange. The xantha-f27 and -f40 are deletions of 14 and 2 bp, respectively, resulting in truncated polypeptides of 1104 and 899 amino acid residues, respectively. Mutation xantha-f41 is an in-frame deletion that removes A439, L440, Q441 and V442 from the resulting protein. Mutation xantha-f58 is most likely a deletion of the whole Xantha-f gene, as no DNA fragments could be detected by PCR or southern blot experiments. The slightly leaky xantha-f60 and non-leaky -f68 mutations each have a missense mutation causing a P393L and G794E exchange in the polypeptide, respectively.     

Analysis of Differential Expression of Barley ESTs during Cold Acclimatization Using Microarray Technology

Abstract: Genomics adds a new dimension to genetic analysis, shifting the focus from the study of a single gene to the whole genome. We have successfully applied the genomics approach based on microarray to the study of genes involved in barley responses to cold stress. About 900 EST clones from barley were obtained from a cDNA library of cold acclimatized leaves of cv. Nure and arrayed, and gene expression analysis done on cold acclimatized vs. control plants. The system allowed for reliable detection of differences in mRNA expression levels, and was confirmed by the finding that numerous previously reported cold-related genes were differentially expressed in treated and untreated plants when evaluated in our arrays. The expression profiles of a sample of genes analysed by the array were confirmed by quantitative RT-PCR.
Previously, identification of novel plant genes was achieved considering a few genes at a time; now many genes can be found as up- or down-regulated based on a one step procedure. Many of the genes we found to be up- or down-regulated do not have an assigned function. This includes 15 of the 78 up-regulated and 8 of the 45 down-regulated clones. Our results add new genes to the group of cold-regulated genes and provide the opportunity to better understand the complex mechanism of stress tolerance.     

Meta-analysis combines affymetrix microarray results across laboratories

With microarray technology becoming more prevalent in recent years, it is now common for several laboratories to employ the same microarray technology to identify differentially expressed genes that are related to the same phenomenon in the same species. Although experimental specifics may be similar, each laboratory will typically produce a slightly different list of statistically significant genes, which calls into question the validity of each gene list (i.e. which list is best). A statistically-based meta-analytic approach to microarray analysis systematically combines results from the different laboratories to provide a single estimate of the degree of differential expression for each gene. This approach provides a more precise view of genes that are of significant interest, while simultaneously allowing for differences between laboratories. The widely-used Affymetrix oligonucleotide array and its software are of particular interest because the results are naturally suited to a meta-analysis. A simulation model based on the Affymetrix platform is developed to examine the adaptive nature of the meta-analytic approach and to illustrate the utility of such an approach in combining microarray results across laboratories.     

Application of z-score transformation to Affymetrix data

Z-score transformation has been successfully used as a normalisation procedure for microarray data generated using radioactively labelled probes with spotted cDNA arrays. One of the advantages of the z-score transformation method is that it provides a way of standardising data across a wide range of experiments and allows the comparison of microarray data independent of the original hybridisation intensities. The feasibility of applying z-score transformation to other types of linear microarray data, specifically that generated using fluorescently labelled probes with Affymetrix chips, was tested in three separate scenarios and is discussed here. In the first scenario, Affymetrix data from the NCBI (National Center for Biotechnology Information) GEO (Gene Expression Omnibus) database was used to demonstrate that z-score transformation preserved the essential phylogenetic grouping between primate species' fibroblast gene expression baseline measurements. The second scenario employed z-score transformation on data consisting of a series of genes spiked-in at known concentrations and arrayed in a Latin square format. We were able to reconstruct the entire set of spike-in concentration curves without prior knowledge of their format by using z-score transformation as the normalisation process. Finally, we show that z-score transformed data maintains the integrity of separate samples from different experiments and laboratories, as demonstrated by accurate grouping of clustered data according to sample identity. We conclude that data normalised by z-score transformation can be easily used with Affymetrix data without noticeable loss of information content. Z-score transformation provides a useful tool for comparisons between experiments and between laboratories that use the Affymetrix platform.     

Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

Background  

Comparison of data produced on different microarray platforms often shows surprising discordance. It is not clear whether this discrepancy is caused by noisy data or by improper probe matching between platforms. We investigated whether the significant level of inconsistency between results produced by alternative gene expression microarray platforms could be reduced by stringent sequence matching of microarray probes. We mapped the short oligo probes of the Affymetrix platform onto cDNA clones of the Stanford microarray platform. Affymetrix probes were reassigned to redefined probe sets if they mapped to the same cDNA clone sequence, regardless of the original manufacturer-defined grouping. The NCI-60 gene expression profiles produced by Affymetrix HuFL platform were recalculated using these redefined probe sets and compared to previously published cDNA measurements of the same panel of RNA samples.     

Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays

C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, 'digital mRNA counting' is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation.     

An integrated cross-platform prognosis study on neuroblastoma patients

There have been several reports about the potential for predicting prognosis of neuroblastoma patients using microarray gene expression profiling of the tumors. However these studies have revealed an apparent diversity in the identity of the genes in their predictive signatures. To test the contribution of the platform to this discrepancy we applied the z-scoring method to minimize the impact of platform and combine gene expression profiles of neuroblastoma (NB) tumors from two different platforms, cDNA and Affymetrix. A total of 12442 genes were common to both cDNA and Affymetrix arrays in our data set. Two-way ANOVA analysis was applied to the combined data set for assessing the relative effect of prognosis and platform on gene expression. We found that 26.6% (3307) of the genes had significant impact on survival. There was no significant impact of microarray platform on expression after application of z-scoring standardization procedure. Artificial neural network (ANN) analysis of the combined data set in a leave-one-out prediction strategy correctly predicted the outcome for 90% of the samples. Hierarchical clustering analysis using the top-ranked 160 genes showed the great separation of two clusters, and the majority of matched samples from the different platforms were clustered next to each other. The ANN classifier trained with our combined cross-platform data for these 160 genes could predict the prognosis of 102 independent test samples with 71% accuracy. Furthermore it correctly predicted the outcome for 85/102 (83%) NB patients through the leave-one-out cross-validation approach. Our study showed that gene expression studies performed in different platforms could be integrated for prognosis analysis after removing variation resulting from different platforms.     

A Cross-Platform Comparison of Affymetrix and Agilent Microarrays Reveals Discordant miRNA Expression in Lung Tumors of c-Raf Transgenic Mice

Non-coding RNAs play major roles in the translational control of gene expression. In order to identify disease-associated miRNAs in precursor lesions of lung cancer, RNA extracts from lungs of either c-Raf transgenic or wild-type (WT) mice were hybridized to the Agilent and Affymetrix miRNA microarray platforms, respectively. This resulted in the detection of a range of miRNAs varying between 111 and 267, depending on the presence or absence of the transgene, on the gender, and on the platform used. Importantly, when the two platforms were compared, only 11–16% of the 586 overlapping genes were commonly detected. With the Agilent microarray, seven miRNAs were identified as significantly regulated, of which three were selectively up-regulated in male transgenic mice. Much to our surprise, when the same samples were analyzed with the Affymetrix platform, only two miRNAs were identified as significantly regulated. Quantitative PCR performed with lung RNA extracts from WT and transgenic mice confirmed only partially the differential expression of significant regulated miRNAs and established that the Agilent platform failed to detect miR-433. Finally, bioinformatic analyses predicted a total of 152 mouse genes as targets of the regulated miRNAs of which 4 and 11 genes were significantly regulated at the mRNA level, respectively in laser micro-dissected lung dysplasia and lung adenocarcinomas of c-Raf transgenic mice. Furthermore, for many of the predicted mouse target genes expression of the coded protein was also repressed in human lung cancer when the publically available database of the Human Protein Atlas was analyzed, thus supporting the clinical significance of our findings. In conclusion, a significant difference in a cross-platform comparison was observed that will have important implications for research into miRNAs.     

Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley

Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the US and Canada. It confers resistance to many, but not all, pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced deletion mutant, showing susceptibility to stem rust pathotype Pgt-MCC, was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 mRNA and protein expression level analyses showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for P. graminis resistance). Genome-wide expression profiling, using the Affymetrix Barley1 GeneChip containing ∼22,840 probe sets, was conducted with Morex and the rpr1 mutant. Of the genes represented on the Barley1 microarray, 20 were up-regulated and 33 were down-regulated by greater than twofold in the mutant, while the Rpg1 mRNA level remained constant. Among the highly down-regulated genes (greater than fourfold), genomic PCR, RT-PCR and Southern analyses identified that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at), were deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and co-segregated with the rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to a mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 GeneChip. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it shows specificity to the Rpg1-mediated resistance pathway.     

Expression profiling by oligonucleotide microarrays spotted on coated polymer slides

We have developed a ready-to-spot polymer microarray slide, which is coated with a uniform layer of reactive electrophilic groups using anthraquinone-mediated photo-coupling chemistry. The slide coating reduces the hydrophobicity of the native polymer significantly, thereby enabling robust and efficient one-step coupling of spotted 5' amino-linked oligonucleotides onto the polymer slide. The utility of the coated polymer slide in gene expression profiling was assessed by fabrication of spotted oligonucleotide microarrays using a collection of 5' amino-linked 70-mer oligonucleotide probes representing 96 yeast genes from Operon. Two-colour hybridizations with labelled cDNA target pools derived from standard grown and heat-shocked wild type yeast cells could reproducibly measure heat shock induced expression of seven different heat shock protein (HSP) genes. Moreover, the observed fold changes were comparable to those reported previously using spotted cDNA arrays and high-density 25-mer oligonucleotide arrays from Affymetrix. The low hybridization signals obtained from the DeltaSSA4 mutant cDNA target, together with the high signal detected in two-colour hybridizations with heat-shocked wild type yeast relative to the DeltaSSA4 mutant strain implies that unspecific binding of cDNA target to the SSA4-specific 70-mer oligonucleotide probes is negligible. Combined, our results indicate that the coated polymer microarray slide represents a robust and cost-effective array platform for pre-spotted oligonucleotide arrays.     

A comparison of cDNA, oligonucleotide, and Affymetrix GeneChip gene expression microarray platforms.

We have conducted a study to compare the variability in measured gene expression levels associated with three types of microarray platforms. Total RNA samples were obtained from liver tissue of four male mice, two each from inbred strains A/J and C57BL/6J. The same four samples were assayed on Affymetrix Mouse Genome Expression Set 430 GeneChips (MOE430A and MOE430B), spotted cDNA microarrays, and spotted oligonucleotide microarrays using eight arrays of each type. Variances associated with measurement error were observed to be comparable across all microarray platforms. The MOE430A GeneChips and cDNA arrays had higher precision across technical replicates than the MOE430B GeneChips and oligonucleotide arrays. The Affymetrix platform showed the greatest range in the magnitude of expression levels followed by the oligonucleotide arrays. We observed good concordance in both estimated expression level and statistical significance of common genes between the Affymetrix MOE430A GeneChip and the oligonucleotide arrays. Despite their apparently high precision, cDNA arrays showed poor concordance with other platforms.     

Metagenome microarray for screening of fosmid clones containing specific genes

A critical step in the process of metagenome analysis is to screen for clones that contain specific genes among a large number of clones. To form one of the sequence-based screening tools of a metagenome library, we designed a format of microarray [metagenome microarray (MGA)] that is arrayed with fosmid library clone DNA samples on a glass slide. We evaluated the MGA using random prime labeled fluorescent probes prepared from PCR products of the target gene and found that we could obtain specific hybridization signals only for the fosmid clone that contained the target gene. We found that the detection limit of the MGA was c. 10 ng microL(-1) of fosmid clone DNA, and that the MGA-based hybridization was quantitative within a concentration range of 10-200 ng microL(-1) of fosmid clone DNA. We used the MGA successfully to identify two fosmid clones that contained 16S rRNA genes from a fosmid library from the sediment of the East Sea, Korea. In conclusion, we have demonstrated that the MGA can be used for screening for fosmid clones containing specific genes in a metagenome library, and that this technology has potential application as a high-throughput metagenome screening tool.