A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.     

Apple juice attenuates genotoxicity and oxidative stress induced by cadmium exposure in multiple organs of rats

The aim of this study was to evaluate the health benefits associated with apple consumption following cadmium exposure. A total of 15 Wistar rats were distributed into three groups (n = 5), as follows: control group (non-treated group, CTRL); cadmium group (Cd) and apple juice group (Cd + AJ). The results showed a decrease in the frequency micronucleated cells in bone marrow and hepatocytes in the group exposed to cadmium and treated with apple juice. Apple juice was also able to reduce the 8OHdG levels and to decrease genetic damage in liver and peripheral blood cells. Catalase (CAT) was decreased following apple juice intake. Taken together, our results demonstrate that apple juice seems to be able to prevent genotoxicity and oxidative stress induced by cadmium exposure in multiple organs of Wistar rats.     

Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium

Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. Oxidative damage and antioxidant properties have been studied in liver and gills of the European bullhead (Cottus gobio) subjected to cadmium (CdCl(2) at nominal concentrations of 0.01 and 1mg/L) for 4 days at either 15°C or 21°C. First, exposure to 1mg Cd/L induced a high mortality rate (67%) in fish held at 21°C. Regarding the antioxidant enzymes, exposure to 0.01 mg Cd/L significantly increased the activity of superoxide dismutase (SOD) and decreased the activity of glutathione reductase (GR) in liver, independently of heat stress. In gills, exposure to 21°C resulted in a significantly increased activity of glutathione peroxidase (GPx), whereas the activity of glutathione S-transferase (GST) was significantly reduced as compared to fish exposed to 15°C. Furthermore, regardless of Cd stress, exposure to elevated temperature resulted in a significant decrease of lipid peroxidation (LPO) level in liver and in a significant increase in the activity of chymotrypsin-like 20S proteasome in both studied tissues of C. gobio. Overall, the present results indicated that elevated temperature and cadmium exposure independently influenced the antioxidant defense system in bullhead with clear tissue-specific and stress-specific antioxidant responses. Further, elevated temperature affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues.     

Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling

Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.     

Molecular analysis of early rice stamen development using organ-specific gene expression profiling

Elucidating the regulatory mechanisms of plant organ formation is an important component of plant developmental biology and will be useful for crop improvement applications. Plant organ formation, or organogenesis, occurs when a group of primordial cells differentiates into an organ, through a well-orchestrated series of events, with a given shape, structure and function. Research over the past two decades has elucidated the molecular mechanisms of organ identity and dorsalventral axis determinations. However, little is known about the molecular mechanisms underlying the successive processes. To develop an effective approach for studying organ formation at the molecular level, we generated organ-specific gene expression profiles (GEPs) reflecting early development in rice stamen. In this study, we demonstrated that the GEPs are highly correlated with early stamen development, suggesting that this analysis is useful for dissecting stamen development regulation. Based on the molecular and morphological correlation, we found that over 26 genes, that were preferentially up-regulated during early stamen development, may participate in stamen development regulation. In addition, we found that differentially expressed genes during early stamen development are clustered into two clades, suggesting that stamen development may comprise of two distinct phases of pattern formation and cellular differentiation. Moreover, the organ-specific quantitative changes in gene expression levels may play a critical role for regulating plant organ formation. Electronic Supplementary Material Supplementary material is available for this article at Xiao-Chun Lu, Hua-Qin Gong contributed equally to this work.     

Molecular classification of scrapie strains in mice using gene expression profiling

Transmissible spongiform encephalopathy strains demonstrate specific prion characteristics, each with specific incubation times, and strain-specific patterns of deposition of the misfolded isoform of prion, PrPSc, in the brains of infected individuals. Different biochemical properties, including glycosylation profiles and the degree of proteinase resistance, have been shown to be strain-specific. However, no relationship between these properties and the phenotypic differences in the subsequent diseases has as yet been determined. Here we explore the utility of gene expression profiles to identify differences in the host response to different strains of prion agent. We identify 114 genes that exhibit significantly different levels of expression in mice infected with three strains of scrapie. These genes represent a pool of genes involved in a strain-specific response to prion disease. We have identified the most discriminatory genes from this list utilizing a wrapper-based feature selection algorithm with external cross-validation.     

Plasma membrane behavior,oxidative damage,and defense mechanism in Phanerochaete chrysosporium under cadmium stress

Microorganisms are essential for maintaining ecosystem balance, and understanding their response to toxic pollutants is important in assessing the potential environmental impacts of such releases. In this study, the response to the heavy metal cadmium and the potential defense or adaptive mechanisms of the widely used white-rot fungus, Phanerochaete chrysosporium, were investigated. The results indicated that cadmium causes plasma membrane damage, including rigidification of lipids, a decrease in H⁺-ATPase activity, and lipid peroxidation. The cellular death may be mediated by oxidative stress with mitochondria membrane potential (MMP) breakdown and reactive oxygen species (ROS) formation. Parts of the cells were able to survive by activating antioxidant defense systems (antioxidant agents and enzymes). Extracellular synthesis of cadmium crystal particles was observed after exposure to dissolved cadmium ion, which is probably another detoxification mechanism in which the dissolved metal is precipitated, thus reducing its bioavailability and toxicity. These physiological responses of P. chrysosporium to cadmium together with the defense mechanisms can provide useful information for the development of fungal-based technologies to reduce the toxic effects of cadmium.     

Antioxidant defense system and cadmium uptake in barley genotypes differing in cadmium tolerance

Using two barley (Hordeum vulgare) cultivars (cvs. Tokak and Hamidiye) nutrient solution experiments were conducted in order to study the genotypic variation in tolerance to Cd toxicity based on (i) development of leaf symptoms, (ii) decreases in dry matter production, (iii) Cd concentration and (iv) changes in antioxidative defense system in leaves (i.e., superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, ascorbic acid and non-protein SH-groups). Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 15, 30, 60 and 120 micromol/L Cd) for different time periods. Of the barley cultivars Hamidiye was particularly sensitive to Cd as judged by the severity and earlier development of Cd toxicity symptoms on leaves. Within 48 h of Cd application Hamidiye rapidly developed severe leaf Cd toxicity symptoms whereas in Tokak the leaf symptoms appeared only slightly. Hamidiye also tended to show more decrease in growth caused by Cd supply when compared to Tokak. The differences in sensitivity to Cd between Tokak and Hamidiye were not related to Cd concentrations in roots and shoots or Cd accumulation per plant. With the exception of catalase, activities of the enzymes involved in detoxification of reactive oxygen species (ROS) were markedly enhanced in Hamidiye by increasing Cd supply. By contrast, in Tokak there was either only a slight increase or no change in the activities of the antioxidative enzymes. Similarly, levels of ascorbic acid and especially non-protein SH-groups were increased in Hamidiye by Cd supply, but not affected in Tokak. The results indicate the existence of a large genotypic variation between barley cultivars for Cd tolerance. The differential Cd tolerance found in the barley cultivars was not related to uptake or accumulation of Cd in plants, indicating importance of internal mechanisms in expression of differential Cd tolerance in barley. As a response to increasing Cd supply particular increases in antioxidative mechanisms in the Cd-sensitive barley cultivar Hamidiye suggest that the high Cd sensitivity of Hamidiye is related to enhanced production and oxidative damage of ROS.