Robustness considerations in selecting efficient two-color microarray designs

The main goal of microarray experiments is to select a small subset of genes that are differentially expressed among competing mRNA samples. For a given set of such mRNA samples, it is possible to consider a number of two-color cDNA microarray designs with a fixed number of arrays. Appropriate criteria can be used to select an efficient design from such a set of alternative experimental designs. In practice, however, microarray expression data often contain missing observations and the most efficient design (with complete observations) for a specific setup may not be efficient in the presence of missing observations. In this article, we propose two criteria to address the robustness of microarray designs against missing observations. We demonstrate the simultaneous use of efficiency and robustness criteria to select good microarray designs for both one-factor and multi-factor experiments. Contact: [email protected]

Optimal factorial designs for cDNA microarray experiments

We consider cDNA microarray experiments when the cell populations have a factorial structure, and investigate the problem of their optimal designing under a baseline parametrization where the objects of interest differ from those under the more common orthogonal parametrization. First, analytical results are given for the $2\times 2$ factorial. Since practical applications often involve a more complex factorial structure, we next explore general factorials and obtain a collection of optimal designs in the saturated, that is, most economic, case. This, in turn, is seen to yield an approach for finding optimal or efficient designs in the practically more important nearly saturated cases. Thereafter, the findings are extended to the more intricate situation where the underlying model incorporates dye-coloring effects, and the role of dye-swapping is critically examined.Comment: Published in at http://dx.doi.org/10.1214/07-AOAS144 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

On the utility of RNA sample pooling to optimize cost and statistical power in RNA sequencing experiments

Background: In gene expression studies, RNA sample pooling is sometimes considered because of budget constraints or lack of sufficient input material. Using microarray technology, RNA sample pooling strategies have been reported to optimize both the cost of data generation as well as the statistical power for differential gene expression (DGE) analysis. For RNA sequencing, with its different quantitative output in terms of counts and tunable dynamic range, the adequacy and empirical validation of RNA sample pooling strategies have not yet been evaluated. In this study, we comprehensively assessed the utility of pooling strategies in RNA-seq experiments using empirical and simulated RNA-seq datasets. Result: The data generating model in pooled experiments is defined mathematically to evaluate the mean and variability of gene expression estimates. The model is further used to examine the trade-off between the statistical power of testing for DGE and the data generating costs. Empirical assessment of pooling strategies is done through analysis of RNA-seq datasets under various pooling and non-pooling experimental settings. Simulation study is also used to rank experimental scenarios with respect to the rate of false and true discoveries in DGE analysis. The results demonstrate that pooling strategies in RNA-seq studies can be both cost-effective and powerful when the number of pools, pool size and sequencing depth are optimally defined. Conclusion: For high within-group gene expression variability, small RNA sample pools are effective to reduce the variability and compensate for the loss of the number of replicates. Unlike the typical cost-saving strategies, such as reducing sequencing depth or number of RNA samples (replicates), an adequate pooling strategy is effective in maintaining the power of testing DGE for genes with low to medium abundance levels, along with a substantial reduction of the total cost of the experiment. In general, pooling RNA samples or pooling RNA samples in conjunction with moderate reduction of the sequencing depth can be good options to optimize the cost and maintain the power

Microarray experiments: Considerations for experimental design

Microarrays are useful tools to investigate the expression of thousands of genes rapidly. Some researchers remain reluctant to use the technology, however, largely because of its expense. Careful design of a microarray experiment is key to generating cost-effective results. This article explores issues that researchers face when embarking on a microarray experiment for the first time. These include decisions about which microarray platform is available for the organism of interest, the degree of replication (biological and technical) needed and which design (direct or indirect, loop or balanced block) is suitable

Developmental biology of wood formation

The wood-forming vascular cambium is responsible for the production of a large part of the biomass on this planet. Yet, there is only limited knowledge on how cell proliferation and differentiation in the cambial meristem are regulated. In this thesis the wood-forming tissues of aspen were used as a model system to identify and characterize molecular factors related to cambial meristem activity. An important regulator of cambial meristem activity is the plant hormone auxin. As polar transport is crucial for the delivery of auxin to the cambial zone, we identified homologues of known regulators of polar auxin transport and described their regulation by environmental and developmental factors. Translating changes in auxin concentration into changes in gene expression involves members of the Aux/IAA gene family. Aspen homologues of Aux/IAA genes were cloned and found to be expressed in a highly tissue-specific fashion, which is further influenced by developmental events and changes in the environment. A major response of trees to environmental changes is the suspension of meristematic growth during winter dormancy. A comparison of gene expression in active and dormant cambia revealed dramatic changes in the transcriptome including the expression of many cold and stress related genes during winter. During the process of wood formation, cells originating in the vascular cambium go through an elaborate process of cell division, cell expansion, secondary wall formation and programmed cell death. Large-scale analysis of gene expression was used to create transcriptional maps of the differentiation process. This extensive dataset allowed us to confirm the proposed functions of various genes involved in wood formation, assign other known genes to specific stages along the developmental gradient and identify a large number of novel potential regulators of wood formation. The data further suggest that the cambial meristem shares regulatory mechanisms with other meristems in addition to its own, specific factors

Analysis of gene expression in female American lobsters (Homarus americanus) to determine reproductive status by oligonucleotide microarray analysis

The American lobster (Homarus americanus) is harvested along the eastern seaboard of North America and forms the economic backbone of many small fishing communities in Atlantic Canada and the northeastern United States. The industry sets minimum size (carapace length) for harvesting American lobsters partially based on determining when females are sexually mature. Given the internal development of ovarian follicles (eggs) protected by a rigid exoskeleton, external assessment of sexual maturity is near impossible. Ovaries can be classified into 7 distinct developmental stages ranging from immature to mature based on a combination of factors including ovary colour, oocyte size, ovary factor, and time of year. Stage 4 ovaries are subdivided into stage 4a (developing) and 4b (mature) ovaries. Ovary stage 4b is important as it represents reproductive commitment. This means that once ovaries progress to stage 4b, they can no longer delay maturation and must continue through until extrusion of eggs onto the pleopods. To stage ovaries and accurately estimate the size at the onset of maturity, female American lobsters must be sacrificed to examine the ovary both grossly and histologically. The discovery of a non-lethal biomarker would result in more accurate ovary staging while allowing the return of the female to the spawning population. Several studies have examined specific reproductive endocrine processes in the American lobster, including the roles of vitellogenin, crustacean hyperglycemic hormone, and gonad-inhibiting hormone. However, no molecular approaches have been attempted to evaluate reproductive status in female American lobsters. This study was the first to examine genome-wide expression changes of reproductive female American lobsters using a novel 14,592 feature, spotted oligonucleotide microarray. The genetic information on the microarray was ~40% functionally annotated based on comparisons with publically available molecular databases. A reference design was used to allow stage specific comparisons between individual ovary stages. Four tissues (ovary, hepatopancreas, eyestalk, and haemocyte pellet) were pooled prior to cDNA synthesis to reduce reagents used and to capture gene expression data from these reproductively important tissues. Therefore, all results were total gene expression in the pooled samples. This study determined 1,774 genes to be statistically significant based on a one-way ANOVA, with 569 functionally annotated genes. The latter were involved in a variety of important biological processes including reproduction, development and growth. A total of 12 genes of interest were chosen for validation using reverse transcription quantitative polymerase chain reaction (RT-qPCR): four vitellogenin genes, two ovary development related proteins, egg-derived tyrosine phosphatase, estradiol-17-β-dehydrogenase 12-B-like, 97 kDa heat shock protein, quaking protein A, growth-hormone inducible transmembrane protein, and inhibitor of growth protein 1-like. Four genes represented distinct copies of vitellogenin, which is converted into vitellin, an important ovary/egg related protein involved in egg yolk. One of these genes was the complete Homarus americanus vitellogenin (HaVg1). A second gene, HaVg2, was 85% similar at a nucleotide level and 74% similar at a protein level with HaVg1. The third EST, given the name HaVg3, was 55% similar at nucleotide and protein levels with HaVg1. The final vitellogenin EST, named HaVg4, was 65% similar at a nucleotide level and 44% similar at a protein level with HaVg1. Three of these vitellogenins (HaVg2, HaVg3, and HaVg4) were unique to this study. The expression profiles of all 4 vitellogenins showed progression from downregulation at stage 2 to upregulation by stage 5, where stages 4a, 4b, and 5 are upregulated. This pattern was consistent between microarrays and RT-qPCR and agreed with previous literature in crustaceans. With the increased anthropogenic and environmental pressures on American lobster stocks, including harvesting and global climate change, this study has only begun to examine potential genetic variations in the American lobster. Future research should examine gene expression on an individual tissue level as genes are regulated on a tissue level. Only 32% of our statistically significant genes were functionally annotated. Further functional annotation may highlight genes associated with reproduction and commitment