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After confirming that long-term serum storage (frozen at -20 C for greater than 3 mo) causes optical density to drift upward, several modifications of an enzyme-linked immunosorbent assay (ELISA) protocol were evaluated to identify a protocol that would reduce background in porcine sera tested for trichinellosis. Modifications evaluated included blocking the antigen-coated ELISA plate with sample diluent containing 10% bovine serum albumin (BSA) or 10% nonfat milk powder (bovine lacto transfer optimizer or BLOTTO), diluting sera in sample diluent containing 10% BSA or 10% BLOTTO, and preincubating samples in sample diluent containing 10% BSA or 10% BLOTTO. Overnight preincubation (approximately 12 hr at 2 C) of fresh sera diluted (1:10) in sample diluent containing 10% BLOTTO significantly reduced background and improved the detection of experimentally infected pigs by enhancing positive-negative discrimination. When testing stored sera, the modified protocol effectively reduced the effect of storage and the kit revealed specificity of 98.4%; there was no loss in sensitivity. The effect of long-term storage at -20 C must therefore be considered when testing swine sera for trichinellosis by ELISA and possibly also when conducting other immunoglobulin assays. The modification described here may prove useful if there is no alternative to using serum stored for greater than 3 mo at -20 C.  相似文献   
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Leek JT  Storey JD 《PLoS genetics》2007,3(9):1724-1735
It has unambiguously been shown that genetic, environmental, demographic, and technical factors may have substantial effects on gene expression levels. In addition to the measured variable(s) of interest, there will tend to be sources of signal due to factors that are unknown, unmeasured, or too complicated to capture through simple models. We show that failing to incorporate these sources of heterogeneity into an analysis can have widespread and detrimental effects on the study. Not only can this reduce power or induce unwanted dependence across genes, but it can also introduce sources of spurious signal to many genes. This phenomenon is true even for well-designed, randomized studies. We introduce "surrogate variable analysis" (SVA) to overcome the problems caused by heterogeneity in expression studies. SVA can be applied in conjunction with standard analysis techniques to accurately capture the relationship between expression and any modeled variables of interest. We apply SVA to disease class, time course, and genetics of gene expression studies. We show that SVA increases the biological accuracy and reproducibility of analyses in genome-wide expression studies.  相似文献   
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Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families.  相似文献   
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Maximal citrate synthase activity (CS) is routinely used as a marker of aerobic capacity and mitochondrial density in skeletal muscle. However, reported CS has been notoriously variable, even with similar experimental protocols and sampling from the same muscles. Exercise training has resulted in increases in CS ranging from 0 to 100%. Previously, it has been reported that acute exercise may significantly affect CS. To investigate the hypothesis that the large variation in CS that occurs with training is influenced by alterations during the exercise itself, we studied CS in human vastus lateralis both in the rested and acutely exercised state while trained and untrained (n = 6). Tissues obtained from four biopsies (untrained rested, untrained acutely exercised, trained rested, and trained acutely exercised) were analyzed spectrophotometrically for maximal CS. Exercise training measured in a rested state resulted in an 18.2% increase in CS (12.3 +/- 0.3 to 14.5 +/- 0.3 micromol x min(-1) x g tissue(-1), P < or = 0.05). However, even greater increases were recorded 1 h after acute exercise: 49.4% in the untrained state (12.3 +/- 0.3 to 18.3 +/- 0.5 micromol x min(-1) x g tissue(-1), P < or = 0.05) and 50.8% in the trained state (14.5 +/- 0.3 to 21.8 +/- 0.4 micromol x min(-1) x g tissue(-1), P < or = 0.05). Ultrastructural analysis, by electron microscopy, supported an effect of acute exercise with the finding of numerous swollen mitochondria 1 h after exercise that may result in greater access to the CS itself in the CS assay. In conclusion, although unexplained, the increased CS with acute exercise can clearly confound training responses and artificially elevate CS values. Therefore, the timing of muscle sampling relative to the last exercise session is critical when measuring CS and offers an explanation for the large variation in CS previously reported.  相似文献   
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It has recently been proposed that variation in DNA methylation at specific genomic locations may play an important role in the development of complex diseases such as cancer. Here, we develop 1- and 2-group multiple testing procedures for identifying and quantifying regions of DNA methylation variability. Our method is the first genome-wide statistical significance calculation for increased or differential variability, as opposed to the traditional approach of testing for mean changes. We apply these procedures to genome-wide methylation data obtained from biological and technical replicates and provide the first statistical proof that variably methylated regions exist and are due to interindividual variation. We also show that differentially variable regions in colon tumor and normal tissue show enrichment of genes regulating gene expression, cell morphogenesis, and development, supporting a biological role for DNA methylation variability in cancer.  相似文献   
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Little is known about the timing of activating memory for objects and their associated perceptual properties, such as colour, and yet this is important for theories of human cognition. We investigated the time course associated with early cognitive processes related to the activation of object shape and object shape+colour representations respectively, during memory retrieval as assessed by repetition priming in an event-related potential (ERP) study. The main findings were as follows: (1) we identified a unique early modulation of mean ERP amplitude during the N1 that was associated with the activation of object shape independently of colour; (2) we also found a subsequent early P2 modulation of mean amplitude over the same electrode clusters associated with the activation of object shape+colour representations; (3) these findings were apparent across both familiar (i.e., correctly coloured – yellow banana) and novel (i.e., incorrectly coloured - blue strawberry) objects; and (4) neither of the modulations of mean ERP amplitude were evident during the P3. Together the findings delineate the timing of object shape and colour memory systems and support the notion that perceptual representations of object shape mediate the retrieval of temporary shape+colour representations for familiar and novel objects.  相似文献   
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