Selection of optimal reference genes for normalization in quantitative RT-PCR

<p>Abstract</p> <p>Background</p> <p>Normalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.</p> <p>Results</p> <p>We propose a robust approach for selecting optimal subset(s) of reference genes with the smallest variance of the corresponding normalizing factors. The normalizing factor variance estimates are based on the estimated unstructured covariance matrix of all available candidate reference genes, adjusting for all possible correlations. Robustness is achieved through bootstrapping all candidate reference gene data and obtaining the bootstrap upper confidence limits for the variances of the log-transformed normalizing factors. The selection of the reference gene subset is optimized with respect to one of the following criteria: (A) to minimize the variability of the normalizing factor; (B) to minimize the number of reference genes with acceptable upper limit on variability of the normalizing factor, (C) to minimize the average rank of the variance of the normalizing factor. The proposed approach evaluates all gene subsets of various sizes rather than ranking individual reference genes by their stability, as in the previous work. In two publicly available data sets and one new data set, our approach identified subset(s) of reference genes with smaller empirical variance of the normalizing factor than in subsets identified using previously published methods. A small simulation study indicated an advantage of the proposed approach in terms of sensitivity to identify the true optimal reference subset in the presence of even modest, especially negative correlation among the candidate reference genes.</p> <p>Conclusions</p> <p>The proposed approach performs comprehensive and robust evaluation of the variability of normalizing factors based on all possible subsets of candidate reference genes. The results of this evaluation provide flexibility to choose from important criteria for selecting the optimal subset(s) of reference genes, unless one subset meets all the criteria. This approach identifies gene subset(s) with smaller variability of normalizing factors than current standard approaches, particularly if there is some nontrivial innate correlation among the candidate genes.</p

Effects of syntactic context on eye movements during reading

Previous research has demonstrated that properties of a currently fixated word and of adjacent words influence eye movement control in reading. In contrast to such local effects, little is known about the global effects on eye movement control, for example global adjustments caused by processing difficulty of previous sentences. In the present study, participants read text passages in which voice (active vs. passive) and sentence structure (embedded vs. non-embedded) were manipulated. These passages were followed by identical target sentences. The results revealed effects of previous sentence structure on gaze durations in the target sentence, implying that syntactic properties of previously read sentences may lead to a global adjustment of eye movement control

The Cultural Project : Formal Chronological Modelling of the Early and Middle Neolithic Sequence in Lower Alsace

Starting from questions about the nature of cultural diversity, this paper examines the pace and tempo of change and the relative importance of continuity and discontinuity. To unravel the cultural project of the past, we apply chronological modelling of radiocarbon dates within a Bayesian statistical framework, to interrogate the Neolithic cultural sequence in Lower Alsace, in the upper Rhine valley, in broad terms from the later sixth to the end of the fifth millennium cal BC. Detailed formal estimates are provided for the long succession of cultural groups, from the early Neolithic Linear Pottery culture (LBK) to the Bischheim Occidental du Rhin Supérieur (BORS) groups at the end of the Middle Neolithic, using seriation and typology of pottery as the starting point in modelling. The rate of ceramic change, as well as frequent shifts in the nature, location and density of settlements, are documented in detail, down to lifetime and generational timescales. This reveals a Neolithic world in Lower Alsace busy with comings and goings, tinkerings and adjustments, and relocations and realignments. A significant hiatus is identified between the end of the LBK and the start of the Hinkelstein group, in the early part of the fifth millennium cal BC. On the basis of modelling of existing dates for other parts of the Rhineland, this appears to be a wider phenomenon, and possible explanations are discussed; full reoccupation of the landscape is only seen in the Grossgartach phase. Radical shifts are also proposed at the end of the Middle Neolithic

Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients

The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5′-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies. © 1999 Cancer Research Campaig

More things in Heaven and Earth: spirit possession, mental disorder, and intentionality

Spirit possession is a common phenomenon around the world in which a non-corporeal agent is involved with a human host. This manifests in a range of maladies or in displacement of the host's agency and identity. Prompted by engagement with the phenomenon in North Africa, this paper draws connections between spirit possession, and the concepts of personhood and intentionality. It employs these concepts to articulate spirit possession, while also developing the intentional stance as formulated by Daniel Dennett. It argues for an understanding of spirit possession as the spirit stance: an intentional strategy that aims at predicting and explaining behaviour by ascribing to an agent (the spirit) beliefs and desires, but is only deployed once the mental states and activity of the subject (the person) fail specific normative distinctions. Applied to behaviours which are generally taken to signal mental disorder, the spirit stance preserves a peculiar form of intentionality where behaviour would otherwise be explained as a consequence of a malfunctioning physical mechanism. Centuries before the modern disciplines of psychoanalysis and phenomenological-psychopathology endeavoured to restore meaning to 'madness', the social institution of spirit possession had been preserving the intentionality of socially deviant behaviour

Fluids and barriers of the CNS establish immune privilege by confining immune surveillance to a two-walled castle moat surrounding the CNS castle

Neuronal activity within the central nervous system (CNS) strictly depends on homeostasis and therefore does not tolerate uncontrolled entry of blood components. It has been generally believed that under normal conditions, the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) prevent immune cell entry into the CNS. This view has recently changed when it was realized that activated T cells are able to breach the BBB and the BCSFB to perform immune surveillance of the CNS. Here we propose that the immune privilege of the CNS is established by the specific morphological architecture of its borders resembling that of a medieval castle. The BBB and the BCSFB serve as the outer walls of the castle, which can be breached by activated immune cells serving as messengers for outside dangers. Having crossed the BBB or the BCSFB they reach the castle moat, namely the cerebrospinal fluid (CSF)-drained leptomeningeal and perivascular spaces of the CNS. Next to the CNS parenchyma, the castle moat is bordered by a second wall, the glia limitans, composed of astrocytic foot processes and a parenchymal basement membrane. Inside the castle, that is the CNS parenchyma proper, the royal family of sensitive neurons resides with their servants, the glial cells. Within the CSF-drained castle moat, macrophages serve as guards collecting all the information from within the castle, which they can present to the immune-surveying T cells. If in their communication with the castle moat macrophages, T cells recognize their specific antigen and see that the royal family is in danger, they will become activated and by opening doors in the outer wall of the castle allow the entry of additional immune cells into the castle moat. From there, immune cells may breach the inner castle wall with the aim to defend the castle inhabitants by eliminating the invading enemy. If the immune response by unknown mechanisms turns against self, that is the castle inhabitants, this may allow for continuous entry of immune cells into the castle and lead to the death of the castle inhabitants, and finally members of the royal family, the neurons. This review will summarize the molecular traffic signals known to allow immune cells to breach the outer and inner walls of the CNS castle moat and will highlight the importance of the CSF-drained castle moat in maintaining immune surveillance and in mounting immune responses in the CNS

Between the Vinča and Linearbandkeramik worlds: the diversity of practices and identities in the 54th–53rd centuries cal BC in south-west Hungary and beyond

Szederk&eacute;ny-Kukorica-dűlő is a large settlement in south-east Transdanubia, Hungary, excavated in advance of road construction, which is notable for its combination of pottery styles, variously including Vinča A, Raži&scaron;te and LBK, and longhouses of a kind otherwise familiar from the LBK world. Formal modelling of its date establishes that the site probably began in the later 54th century cal BC, lasting until the first decades of the 52nd century cal BC. Occupation, featuring longhouses, pits and graves, probably began at the same time on the east and west parts of the settlement, the central part starting a decade or two later; the western part was probably abandoned last. Vinča pottery is predominantly associated with the east and central parts of the site, and Raži&scaron;te pottery with the west. Formal modelling of the early history and diaspora of longhouses in the LBK world suggests their emergence in the Formative LBK of Transdanubia c. 5500 cal BC and then rapid diaspora in the middle of the 54th century cal BC, associated with the &lsquo;earliest&rsquo; (&auml;lteste) LBK. The adoption of longhouses at Szederk&eacute;ny thus appears to come a few generations after the start of the diaspora. Rather than explaining the mixture of things, practices and perhaps people at Szederk&eacute;ny by reference to problematic notions such as hybridity, we propose instead a more fluid and varied vocabulary including combination and amalgamation, relationships and performance in the flow of social life, and networks; this makes greater allowance for diversity and interleaving in a context of rapid change