76 research outputs found
Shape-shifting and pushing against the odds: staff perceptions of the experiences of first generation students in South Africa and the UK
The principles of diversity and inclusion are valued across the higher education sector, but the ways in which these principles are translated into pedagogic practice are not always evident. Students who are first in their family to attend university continue to report barriers to full participation in university life. They are more likely to leave their studies early, and to achieve lower grades in their final qualifications, than students whose families have previous experience of higher education. The purpose of this study was to explore whether a mismatch between staff perceptions and students’ experiences might be a possible contributor to these disparities. The study explored and compared staff discourses about the experiences of first generation students at two universities, one in the United Kingdom (UK), and the other in South Africa (SA). One-to-one interviews were carried out with 40 staff members (20 at each institution) to explore their views about first generation students. The results showed that staff were well aware of challenges faced by first generation students; however, they were unsure of their roles in relation to shaping an inclusive environment, and tended not to consider how to use the assets that they believed first generation students bring with them to higher education. This paper explores these staff discourses; and considers proposals for challenging commonly-voiced assumptions about students and university life in a broader context of diversity and inclusive teaching practice
SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.
Characterization of whole blood gene expression profiles as a sequel to globin mRNA reduction in patients with sickle cell disease
Global transcriptome analysis of whole blood RNA using microarrays has been proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA. This is a particular problem in patients with sickle cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation. In order to accurately measure the steady state blood transcriptome in sickle cell patients we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA for genome-wide transcriptome analyses using microarrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle cell disease patients. This led to an improvement in microarray data quality by reducing data variability, with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. Analysis of differences between the whole blood transcriptome and PBMC transcriptome revealed important erythrocyte genes that participate in sickle cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases
Fluorocarbon Contamination from the Drill on the Mars Science Laboratory: Potential Science Impact on Detecting Martian Organics by Sample Analysis at Mars (SAM)
Polytetrafluoroethylene (PTFE or trade name: Teflon by Dupont Co.) has been detected in rocks drilled during terrestrial testing of the Mars Science Laboratory (MSL) drilling hardware. The PTFE in sediments is a wear product of the seals used in the Drill Bit Assemblies (DBAs). It is expected that the drill assembly on the MSL flight model will also shed Teflon particles into drilled samples. One of the primary goals of the Sample Analysis at Mars (SAM) instrument suite on MSL is to test for the presence of martian organics in samples. Complications introduced by the potential presence of PTFE in drilled samples to the SAM evolved gas analysis (EGA or pyrolysisquadrupole mass spectrometry, pyr-QMS) and pyrolysis- gas chromatography mass spectrometry (Pyr- GCMS) experiments was investigated
Comparison of Whole Blood and Peripheral Blood Mononuclear Cell Gene Expression for Evaluation of the Perioperative Inflammatory Response in Patients with Advanced Heart Failure
Background: Heart failure (HF) prevalence is increasing in the United States.
Mechanical Circulatory Support (MCS) therapy is an option for Advanced HF
(AdHF) patients. Perioperatively, multiorgan dysfunction (MOD) is linked to the
effects of device implantation, augmented by preexisting HF. Early recognition of
MOD allows for better diagnosis, treatment, and risk prediction. Gene expression
profiling (GEP) was used to evaluate clinical phenotypes of peripheral blood
mononuclear cells (PBMC) transcriptomes obtained from patients’ blood samples.
Whole blood (WB) samples are clinically more feasible, but their performance in
comparison to PBMC samples has not been determined.
Methods: We collected blood samples from 31 HF patients (57¡15 years old)
undergoing cardiothoracic surgery and 7 healthy age-matched controls, between
2010 and 2011, at a single institution. WB and PBMC samples were collected at a
single timepoint postoperatively (median day 8 postoperatively) (25–75% IQR 7–14
days) and subjected to Illumina single color Human BeadChip HT12 v4 whole
genome expression array analysis. The Sequential Organ Failure Assessment
(SOFA) score was used to characterize the severity of MOD into low (# 4 points),
intermediate (5–11), and high ($ 12) risk categories correlating with GEP.
Results: Results indicate that the direction of change in GEP of individuals with
MOD as compared to controls is similar when determined from PBMC versus WB.
The main enriched terms by Gene Ontology (GO) analysis included those involved
in the inflammatory response, apoptosis, and other stress response related
pathways. The data revealed 35 significant GO categories and 26 pathways
overlapping between PBMC and WB. Additionally, class prediction using machine
learning tools demonstrated that the subset of significant genes shared by PBMC
and WB are sufficient to train as a predictor separating the SOFA groups.
Conclusion: GEP analysis of WB has the potential to become a clinical tool for
immune-monitoring in patients with MO
Detection of Organic Constituents Including Chloromethylpropene in the Analyses of the ROCKNEST Drift by Sample Analysis at Mars (SAM)
key challenge in assessing the habitability of martian environments is the detection of organic matter - a requirement of all life as we know it. The Curiosity rover, which landed on August 6, 2012 in Gale Crater of Mars, includes the Sample Analysis at Mars (SAM) instrument suite capable of in situ analysis of gaseous organic components thermally evolved from sediment samples collected, sieved, and delivered by the MSL rover. On Sol 94, SAM received its first solid sample: scooped sediment from Rocknest that was sieved to <150 m particle size. Multiple 10-40 mg portions of the scoop #5 sample were delivered to SAM for analyses. Prior to their introduction, a blank (empty cup) analysis was performed. This blank served 1) to clean the analytical instrument of SAMinternal materials that accumulated in the gas processing system since integration into the rover, and 2) to characterize the background signatures of SAM. Both the blank and the Rocknest samples showed the presence of hydrocarbon components
An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA
<p>Abstract</p> <p>Background</p> <p>Validation of microarrays data by quantitative real-time PCR (qPCR) is often limited by the low amount of available RNA. This raised the possibility to perform validation experiments on the amplified amino allyl labeled RNA (AA-aRNA) leftover from microarrays. To test this possibility, we used an ongoing study of our laboratory aiming at identifying new biomarkers of graft rejection by the transcriptomic analysis of blood cells from brain-dead organ donors.</p> <p>Results</p> <p>qPCR for ACTB performed on AA-aRNA from 15 donors provided Cq values 8 cycles higher than when original RNA was used (P < 0.001), suggesting a strong inhibition of qPCR performed on AA-aRNA. When expression levels of 5 other genes were measured in AA-aRNA generated from a universal reference RNA, qPCR sensitivity and efficiency were decreased. This prevented the quantification of one low-abundant gene, which was readily quantified in un-amplified and un-labeled RNA. To overcome this limitation, we modified the reverse transcription (RT) protocol that generates cDNA from AA-aRNA as follows: addition of a denaturation step and 2-min incubation at room temperature to improve random primers annealing, a transcription initiation step to improve RT, and a final treatment with RNase H to degrade remaining RNA. Tested on universal reference AA-aRNA, these modifications provided a gain of 3.4 Cq (average from 5 genes, P < 0.001) and an increase of qPCR efficiency (from -1.96 to -2.88; P = 0.02). They also allowed for the detection of a low-abundant gene that was previously undetectable. Tested on AA-aRNA from 15 brain-dead organ donors, RT optimization provided a gain of 2.7 cycles (average from 7 genes, P = 0.004). Finally, qPCR results significantly correlated with microarrays.</p> <p>Conclusion</p> <p>We present here an optimized RT protocol for validation of microarrays by qPCR from AA-aRNA. This is particularly valuable in experiments where limited amount of RNA is available.</p
Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification
BACKGROUND: Microarray-based gene expression profiling is a powerful approach for the identification of molecular biomarkers of disease, particularly in human cancers. Utility of this approach to measure responses to therapy is less well established, in part due to challenges in obtaining serial biopsies. Identification of suitable surrogate tissues will help minimize limitations imposed by those challenges. This study describes an approach used to identify gene expression changes that might serve as surrogate biomarkers of drug activity. METHODS: Expression profiling using microarrays was applied to peripheral blood mononuclear cell (PBMC) samples obtained from patients with advanced colorectal cancer participating in a Phase III clinical trial. The PBMC samples were harvested pre-treatment and at the end of the first 6-week cycle from patients receiving standard of care chemotherapy or standard of care plus SU5416, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitor. Results from matched pairs of PBMC samples from 23 patients were queried for expression changes that consistently correlated with SU5416 administration. RESULTS: Thirteen transcripts met this selection criterion; six were further tested by quantitative RT-PCR analysis of 62 additional samples from this trial and a second SU5416 Phase III trial of similar design. This method confirmed four of these transcripts (CD24, lactoferrin, lipocalin 2, and MMP-9) as potential biomarkers of drug treatment. Discriminant analysis showed that expression profiles of these 4 transcripts could be used to classify patients by treatment arm in a predictive fashion. CONCLUSIONS: These results establish a foundation for the further exploration of peripheral blood cells as a surrogate system for biomarker analyses in clinical oncology studies
A Practical Platform for Blood Biomarker Study by Using Global Gene Expression Profiling of Peripheral Whole Blood
Background: Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study. Methods and Findings: We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling. We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal. Conclusion: We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study
Investigation of Variation in Gene Expression Profiling of Human Blood by Extended Principle Component Analysis
BACKGROUND: Human peripheral blood is a promising material for biomedical research. However, various kinds of biological and technological factors result in a large degree of variation in blood gene expression profiles. METHODOLOGY/PRINCIPAL FINDINGS: Human peripheral blood samples were drawn from healthy volunteers and analysed using the Human Genome U133Plus2 Microarray. We applied a novel approach using the Principle Component Analysis and Eigen-R(2) methods to dissect the overall variation of blood gene expression profiles with respect to the interested biological and technological factors. The results indicated that the predominating sources of the variation could be traced to the individual heterogeneity of the relative proportions of different blood cell types (leukocyte subsets and erythrocytes). The physiological factors like age, gender and BMI were demonstrated to be associated with 5.3% to 9.2% of the total variation in the blood gene expression profiles. We investigated the gene expression profiles of samples from the same donors but with different levels of RNA quality. Although the proportion of variation associated to the RNA Integrity Number was mild (2.1%), the significant impact of RNA quality on the expression of individual genes was observed. CONCLUSIONS: By characterizing the major sources of variation in blood gene expression profiles, such variability can be minimized by modifications to study designs. Increasing sample size, balancing confounding factors between study groups, using rigorous selection criteria for sample quality, and well controlled experimental processes will significantly improve the accuracy and reproducibility of blood transcriptome study
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