RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

<p>Abstract</p> <p>Background</p> <p>Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.</p> <p>Results</p> <p>Comparing the methods pair-wise, Manual <it>vs</it>. Ratio showed concordance (good <it>vs</it>. degraded RNA) in 20/24, Manual <it>vs</it>. RIN in 23/24, and Ratio <it>vs</it>. RIN in 21/24 samples. All three methods were concordant in 20/24 samples. The comparison between RNA quality and gene expression analysis showed that pieces from the same tumor and with good RNA quality clustered together in most cases, whereas those with poor quality often clustered apart. The number of samples clustering in an unexpected manner was lower for the Manual (n = 1) and RIN methods (n = 2) as compared to the Ratio method (n = 5).</p> <p>Assigning the data into two groups, RIN ≥ 6 or RIN < 6, all but one of the top ten differentially expressed genes showed decreased expression in the latter group; <it>i.e</it>. when the RNA became degraded. Ontological mapping using GoMiner (p ≤ 0.05; ≥ 3 genes changed) revealed deoxyribonuclease activity, collagen, regulation of cell adhesion, cytosolic ribosome, and NADH dehydrogenase activity, to be the five categories most affected by RNA quality.</p> <p>Conclusion</p> <p>The results indicate that the Manual and RIN methods are superior to the Ratio method for evaluating RNA quality in fresh frozen breast cancer tissues. The objective measurement when using the RIN method is an advantage. Furthermore, the inclusion of samples with degraded RNA may profoundly affect gene expression levels.</p

Kognitive utfordringer i to norske lærebokserier fra ungdomsskolen – en mixed methods studie

I denne masteroppgaven i matematikkdidaktikk undersøker vi de kognitive utfordringene i oppgavene i to norske lærebokserier for ungdomsskolen. Norske matematikklærere benytter seg i stor grad av læreboka i undervisningen (Mullis, Martin, Foy & Arora, 2012), samt at en rekke studier hevder at det er læreboka og oppgavene i dem som i stor grad legger til rette for hva elevene lærer seg (Fan, Miao & Zhu, 2013; Hiebert m.fl., 1997; Hiebert & Wearne, 1997; Pepin & Haggarty, 2007; Robitaille & Travers, 1992). På bakgrunn av dette ønsker vi å undersøke denne problemstillingen: I hvilken grad får elevene kognitive utfordringer gjennom oppgavene som gis i de to mest brukte lærebøkene på ungdomskolen i Norge? 1. Hvilken grad av kognitive nivåkrav krever oppgavene? 2. Hvilke typer svar krever oppgavene? For å svare på denne problemstillingen har vi gjennomført en mixed methods studie. Analysen er delt inn i en horisontal del og en vertikal del. Den horisontale analysen har gitt oss oversikt over bakgrunnsinformasjonen og en generell oversikt over læreverkene vi har benyttet oss av. Denne analysen har blant annet gitt oss grunnlaget for å dele oppgavene inn i ulike tema. Den vertikale analysen gikk mer i dybden. Her benyttet vi oss av to ulike tilnærminger, en kvantitativ og en kvalitativ del. I den kvantitative analysen kodet vi oppgaver i forhold til vårt konseptuelle rammeverk. I tillegg gjennomførte vi en kvalitativ tilleggsanalyse av et utvalg av lærebøkenes grubleoppgaver. Den kvantitative analysen av oppgavene har hovedtyngden i denne masteroppgaven, mens hensikten med den kvalitative tilleggsanalysen var å ytterligere belyse elementer som vårt konseptuelle rammeverk ikke fanget opp. Funnene våre viser at oppgavene i begge lærebokseriene i stor grad er lavere kognitivt krevende. Særlig Faktor, men også Maximum gir elevene få muligheter til å møte på kognitivt utfordrende oppgaver. Oppgaver med lavere kognitive nivåkrav gir elevene minimal mulighet for å møte på kognitive utfordringer som igjen fører til svakere utvikling av matematisk kompetanse. Flesteparten av oppgavene vi har kategorisert krever kun at elevene gir et svar, i stedet for en forklaring eller en begrunnelse. I og med at en så stor andel av oppgavene kun krever et svar mener vi dette begrenser de kognitive utfordringene i oppgavene

Genotyping of human papillomavirus in triaging of low-grade cervical cytology.

OBJECTIVE: The objective of the study was to evaluate whether typing of human papillomavirus (HPV) among women with low-grade cervical cytology can improve the ability to identify women with cervical cancer or cervical intraepithelial neoplasia grade III (CIN III or worse). STUDY DESIGN: A total of 1595 women with low-grade cervical cytology participating in a randomized implementation trial of HPV triaging using Hybrid Capture II were also HPV genotyped and CIN III or worse predictive values evaluated. RESULTS: HPV 16 was detected in 57% of cases with CIN III or worse but only among 24% of all tested women. Testing for the 3 HPV types with highest risk (HPV16/31/33) detected 77% of CIN III or worse, with 36% of women testing positive. Positivity for the other high-risk HPV types had a decreased risk for CIN III or worse. CONCLUSION: Different high-risk HPV types confer different risks for the presence of CIN III or worse, implying that HPV genotyping could be useful for the optimization of triaging strategies

Impact of an icy winter on the Pacific oyster (Crassostrea gigas Thunberg, 1793) populations in Scandinavia

The Pacific oyster (Crassostrea gigas) is an invasive species that has dispersed into Scandinavia during the last few decades. The objective of this study was to evaluate the effects of extreme winter conditions on the mortality of the Pacific oyster in Scandinavia. The study was done by compiling mortality data from independent surveys in Denmark, Sweden and Norway. Winter mortality of the oysters increased with latitude, which can be explained by the colder climate experienced at higher latitudes. Mortality was also found to be affected by site specific conditions such as water depth at the sampling sites of oyster populations. Despite the severe winter conditions of 2009/2010 causing high mortality, the Pacific oyster still exists in large numbers in Scandinavia. The present investigation indicates that extreme winter onditions may result in a temporary reduction of the density of the Pacific oyster, but that the species can be expected to continue its invasion of Scandinavian coastal areas.publishedVersio

Gene expression profiling in primary breast cancer distinguishes patients developing local recurrence after breast-conservation surgery, with or without postoperative radiotherapy

Introduction Some patients with breast cancer develop local recurrence after breast-conservation surgery despite postoperative radiotherapy, whereas others remain free of local recurrence even in the absence of radiotherapy. As clinical parameters are insufficient for identifying these two groups of patients, we investigated whether gene expression profiling would add further information. Methods We performed gene expression analysis (oligonucleotide arrays, 26,824 reporters) on 143 patients with lymph node-negative disease and tumor-free margins. A support vector machine was employed to build classifiers using leave-one-out cross-validation. Results Within the estrogen receptor-positive (ER+) subgroup, the gene expression profile clearly distinguished patients with local recurrence after radiotherapy (n = 20) from those without local recurrence (n = 80 with or without radiotherapy). The receiver operating characteristic (ROC) area was 0.91, and 5,237 of 26,824 reporters had a P value of less than 0.001 (false discovery rate = 0.005). This gene expression profile provides substantially added value to conventional clinical markers (for example, age, histological grade, and tumor size) in predicting local recurrence despite radiotherapy. Within the ER- subgroup, a weaker, but still significant, signal was found (ROC area = 0.74). The ROC area for distinguishing patients who develop local recurrence from those who remain local recurrence-free in the absence of radiotherapy was 0.66 (combined ER+/ER-). Conclusion A highly distinct gene expression profile for patients developing local recurrence after breast-conservation surgery despite radiotherapy has been identified. If verified in further studies, this profile might be a most important tool in the decision making for surgery and adjuvant therapy

Khoe-San genomes reveal unique variation and confirm the deepest population divergence in Homo sapiens

Abstract: The southern African indigenous Khoe-San populations harbor the most divergent lineages of all living peoples. Exploring their genomes is key to understanding deep human history. We sequenced 25 full genomes from five Khoe-San populations, revealing many novel variants, that 25% of variants are unique to the Khoe-San, and that the Khoe-San group harbors the greatest level of diversity across the globe. In line with previous studies, we found several gene regions with extreme values in genome-wide scans for selection, potentially caused by natural selection in the lineage leading to Homo sapiens and more recent in time. These gene regions included immunity-, sperm-, brain-, diet-, and muscle-related genes. When accounting for recent admixture, all Khoe-San groups display genetic diversity approaching the levels in other African groups and a reduction in effective population size starting around 100,000 years ago. Hence, all human groups show a reduction in effective population size commencing around the time of the Out-of- Africa migrations, which coincides with changes in the paleoclimate records, changes that potentially impacted all humans at the time

External validation of the RISC, RISC-Malawi, and PERCH clinical prediction rules to identify risk of death in children hospitalized with pneumonia

From Crossref journal articles via Jisc Publications RouterBackground Existing scores to identify children at risk of hospitalized pneumonia-related mortality lack broad external validation. Our objective was to externally validate three such risk scores. Methods We applied the Respiratory Index of Severity in Children (RISC) for HIV-negative children, the RISC-Malawi, and the Pneumonia Etiology Research for Child Health (PERCH) scores to hospitalized children in the Pneumonia REsearch Partnerships to Assess WHO REcommendations (PREPARE) data set. The PREPARE data set includes pooled data from 41 studies on pediatric pneumonia from across the world. We calculated test characteristics and the area under the curve (AUC) for each of these clinical prediction rules. Results The RISC score for HIV-negative children was applied to 3574 children 0-24 months and demonstrated poor discriminatory ability (AUC = 0.66, 95% confidence interval (CI) = 0.58-0.73) in the identification of children at risk of hospitalized pneumonia-related mortality. The RISC-Malawi score had fair discriminatory value (AUC = 0.75, 95% CI = 0.74-0.77) among 17 864 children 2-59 months. The PERCH score was applied to 732 children 1-59 months and also demonstrated poor discriminatory value (AUC = 0.55, 95% CI = 0.37-0.73). Conclusions In a large external application of the RISC, RISC-Malawi, and PERCH scores, a substantial number of children were misclassified for their risk of hospitalized pneumonia-related mortality. Although pneumonia risk scores have performed well among the cohorts in which they were derived, their performance diminished when externally applied. A generalizable risk assessment tool with higher sensitivity and specificity to identify children at risk of hospitalized pneumonia-related mortality may be needed. Such a generalizable risk assessment tool would need context-specific validation prior to implementation in that setting.11pubpub

Derivation and validation of a novel risk assessment tool to identify children aged 2-59 months at risk of hospitalised pneumonia-related mortality in 20 countries

INTRODUCTION: Existing risk assessment tools to identify children at risk of hospitalised pneumonia-related mortality have shown suboptimal discriminatory value during external validation. Our objective was to derive and validate a novel risk assessment tool to identify children aged 2-59 months at risk of hospitalised pneumonia-related mortality across various settings. METHODS: We used primary, baseline, patient-level data from 11 studies, including children evaluated for pneumonia in 20 low-income and middle-income countries. Patients with complete data were included in a logistic regression model to assess the association of candidate variables with the outcome hospitalised pneumonia-related mortality. Adjusted log coefficients were calculated for each candidate variable and assigned weighted points to derive the Pneumonia Research Partnership to Assess WHO Recommendations (PREPARE) risk assessment tool. We used bootstrapped selection with 200 repetitions to internally validate the PREPARE risk assessment tool. RESULTS: A total of 27 388 children were included in the analysis (mean age 14.0 months, pneumonia-related case fatality ratio 3.1%). The PREPARE risk assessment tool included patient age, sex, weight-for-age z-score, body temperature, respiratory rate, unconsciousness or decreased level of consciousness, convulsions, cyanosis and hypoxaemia at baseline. The PREPARE risk assessment tool had good discriminatory value when internally validated (area under the curve 0.83, 95% CI 0.81 to 0.84). CONCLUSIONS: The PREPARE risk assessment tool had good discriminatory ability for identifying children at risk of hospitalised pneumonia-related mortality in a large, geographically diverse dataset. After external validation, this tool may be implemented in various settings to identify children at risk of hospitalised pneumonia-related mortality