Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) have critical functions in various biological processes. MiRNA profiling is an important tool for the identification of differentially expressed miRNAs in normal cellular and disease processes. A technical challenge remains for high-throughput miRNA expression analysis as the number of miRNAs continues to increase with <it>in silico </it>prediction and experimental verification. Our study critically evaluated the performance of a novel miRNA expression profiling approach, quantitative RT-PCR array (qPCR-array), compared to miRNA detection with oligonucleotide microchip (microarray).</p> <p>Results</p> <p>High reproducibility with qPCR-array was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array.</p> <p>Conclusion</p> <p>Our studies demonstrated high reproducibility of TaqMan qPCR-array. Comparison between different reverse transcription reactions and qPCR-arrays performed on different days indicated that reverse transcription reactions did not introduce significant variation in the results. The use of cDNA pre-amplification increased the sensitivity of miRNA detection. Although there was variability associated with pre-amplification in low abundance miRNAs, the latter did not involve any systemic bias in the estimation of miRNA expression. Comparison between microarray and qPCR-array indicated superior sensitivity and specificity of qPCR-array.</p

Altered Macrophage Phenotype Transition Impairs Skeletal Muscle Regeneration

Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11b-diphtheria toxin receptor transgenic mice to transiently deplete monocytes/macrophages at multiple stages before and after muscle injury induced by cardiotoxin. Fat accumulation within regenerated muscle was maximal when ablation occurred at the same time as cardiotoxin-induced injury. Early ablation (day 1 after cardiotoxin) resulted in the smallest regenerated myofiber size together with increased residual necrotic myofibers and fat accumulation. However, muscle regeneration after late (day 4) ablation was similar to controls. Levels of inflammatory cells in injured muscle following early ablation and associated with impaired muscle regeneration were determined by flow cytometry. Delayed, but exaggerated, monocyte [CD11b+(CD90/B220/CD49b/NK1.1/Ly6G)−(F4/80/I-Ab/CD11c)−Ly6C+/−] accumulation occurred; interestingly, Ly6C+ and Ly6C− monocytes were present concurrently in ablated animals and control mice. In addition to monocytes, proinflammatory, Ly6C+ macrophage accumulation following early ablation was delayed compared to controls. In both groups, CD11b+F4/80+ cells exhibited minimal expression of the M2 markers CD206 and CD301. Nevertheless, early ablation delayed and decreased the transient accumulation of CD11b+F4/80+Ly6C−CD301− macrophages; in control animals, the later tissue accumulation of these cells appeared to correspond to that of anti-inflammatory macrophages, determined by cytokine production and arginase activity. In summary, impairments in muscle regeneration were associated with exaggerated monocyte recruitment and reduced Ly6C− macrophages; the switch of macrophage/monocyte subsets is critical to muscle regeneration

Clinical and Translational Scientist Career Success: Metrics for Evaluation

Despite the increased emphasis on formal training in clinical and translational research and the growth in the number and scope of training programs over the past decade, the impact of training on research productivity and career success has yet to be fully evaluated at the institutional level. In this article, the Education Evaluation Working Group of the Clinical and Translational Science Award Consortium introduces selected metrics and methods associated with the assessment of key factors that affect research career success. The goals in providing this information are to encourage more consistent data collection across training sites, to foster more rigorous and systematic exploration of factors associated with career success, and to help address previously identified difficulties in program evaluation

Inclusion of postdoctoral trainees in a translational science training TL1 program was associated with greater diversification of research across the translational science continuum: a bibliometric analysis of TL1 trainee publications

The NIH National Center for Advancing Translational Science (NCATS) was established to support translational research that spans the entire TS Continuum, with the goal of bridging the gap between preclinical biomedical research and real-world applications to advance treatments to patients more quickly. In 2018, the Translational Science Training (TST) TL1 Program at the University of Texas Health Science Center at San Antonio implemented new strategies to better include and encourage research more broadly across the TS Continuum, including the addition of postdoctoral scientists and a clinically trained Program Co-Director, expansion of team science and community engagement programming, and targeted trainee recruitment from schools of nursing, dentistry, and allied health, in addition to medicine. The objective of this bibliometric analysis was to determine if the program exhibited a more diverse mix of T-types after the adjustments made in 2018. The TST/TL1 Program experienced a shift in T-type, from mostly T0 (preclinical) to more T3/T4 (clinical implementation/public health) research, after new strategies were implemented. This supports the conclusion that strategic programmatic adjustments by an NCATS-funded predoctoral training program resulted in outcomes that better align with NCATS priorities to develop Trainees who contribute across the entire TS Continuum

Multifactorial Analysis of Differences Between Sporadic Breast Cancers and Cancers Involving BRCA1 and BRCA2 Mutations

Background: We have previously demonstrated that breast cancers associated with inherited BRCA1 and BRCA2 gene mutations differ from each other in their histopathologic appearances and that each of these types differs from breast cancers in patients unselected for family history (i.e., sporadic cancers). We have now conducted a more detailed examination of cytologic and architectural features of these tumors. Methods: Specimens of tumor tissue (5-µm-thick sections) were examined independently by two pathologists, who were unaware of the case or control subject status, for the presence of cell mitosis, lymphocytic infiltration, continuous pushing margins, and solid sheets of cancer cells; cell nuclei, cell nucleoli, cell necrosis, and cell borders were also evaluated. The resulting data were combined with previously available information on tumor type and tumor grade and further evaluated by multifactorial analysis. All statistical tests are two-sided. Results: Cancers associated with BRCA1 mutations exhibited higher mitotic counts (P = .001), a greater proportion of the tumor with a continuous pushing margin (P<.0001), and more lymphocytic infiltration (P = .002) than sporadic (i.e., control) cancers. Cancers associated with BRCA2 mutations exhibited a higher score for tubule formation (fewer tubules) (P = .0002), a higher proportion of the tumor perimeter with a continuous pushing margin (P<.0001), and a lower mitotic count (P = .003) than control cancers. Conclusions: Our study has identified key features of the histologic phenotypes of breast cancers in carriers of mutant BRCA1 and BRCA2 genes. This information may improve the classification of breast cancers in individuals with a family history of the disease and may ultimately aid in the clinical management of patients. [J Natl Cancer Inst 1998;90:1138-45

Mammalian microRNAs: a small world for fine-tuning gene expression

The basis of eukaryotic complexity is an intricate genetic architecture where parallel systems are involved in tuning gene expression, via RNA-DNA, RNA-RNA, RNA-protein, and DNA-protein interactions. In higher organisms, about 97% of the transcriptional output is represented by noncoding RNA (ncRNA) encompassing not only rRNA, tRNA, introns, 5′ and 3′ untranslated regions, transposable elements, and intergenic regions, but also a large, rapidly emerging family named microRNAs. MicroRNAs are short 20-22-nucleotide RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. MicroRNAs are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the cytoplasmic Dicer, a member of the RNase III enzyme family. A recent analysis of the genomic location of human microRNA genes suggested that 50% of microRNA genes are located in cancer-associated genomic regions or in fragile sites. This review focuses on the possible implications of microRNAs in post-transcriptional gene regulation in mammalian diseases, with particular focus on cancer. We argue that developing mouse models for deleted and/or overexpressed microRNAs will be of invaluable interest to decipher the regulatory networks where microRNAs are involved

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing

International audienceCurrent sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the world's oceans

Strategies to improve control of sexually transmissible infections in remote Australian Aboriginal communities: a stepped-wedge, cluster-randomised trial

BACKGROUND: Remote Australian Aboriginal communities have among the highest diagnosed rates of sexually transmissible infections (STIs) in the world. We did a trial to assess whether continuous improvement strategies related to sexual health could reduce infection rates. METHODS: In this stepped-wedge, cluster-randomised trial (STIs in remote communities: improved and enhanced primary health care [STRIVE]), we recruited primary health-care centres serving Aboriginal communities in remote areas of Australia. Communities were eligible to participate if they were classified as very remote, had a population predominantly of Aboriginal people, and only had one primary health-care centre serving the population. The health-care centres were grouped into clusters on the basis of geographical proximity to each other, population size, and Aboriginal cultural ties including language connections. Clusters were randomly assigned into three blocks (year 1, year 2, and year 3 clusters) using a computer-generated randomisation algorithm, with minimisation to balance geographical region, population size, and baseline STI testing level. Each year for 3 years, one block of clusters was transitioned into the intervention phase, while those not transitioned continued usual care (control clusters). The intervention phase comprised cycles of reviewing clinical data and modifying systems to support improved STI clinical practice. All investigators and participants were unmasked to the intervention. Primary endpoints were community prevalence and testing coverage in residents aged 16–34 years for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis . We used Poisson regression analyses on the final dataset and compared STI prevalences and testing coverage between control and intervention clusters. All analyses were by intention to treat and models were adjusted for time as an independent covariate in overall analyses. This study was registered with the Australia and New Zealand Clinical Trials Registry, ACTRN12610000358044. FINDINGS: Between April, 2010, and April, 2011, we recruited 68 primary care centres and grouped them into 24 clusters, which were randomly assigned into year 1 clusters (estimated population aged 16–34 years, n=11 286), year 2 clusters (n=10 288), or year 3 clusters (n=13 304). One primary health-care centre withdrew from the study due to restricted capacity to participate. We detected no difference in the relative prevalence of STIs between intervention and control clusters (adjusted relative risk [RR] 0·97, 95% CI 0·84–1·12; p=0·66). However, testing coverage was substantially higher in intervention clusters (22%) than in control clusters (16%; RR 1·38; 95% CI 1·15–1·65; p=0·0006). INTERPRETATION: Our intervention increased STI testing coverage but did not have an effect on prevalence. Additional interventions that will provide increased access to both testing and treatment are required to reduce persistently high prevalences of STIs in remote communities.James Ward, Rebecca J Guy, Alice R Rumbold, Skye McGregor, Handan Wand, Hamish McManus, Amalie Dyda, Linda Garton, Belinda Hengel, Bronwyn J Silver, Debbie Taylor-Thomson, Janet Knox, Basil Donovan, Matthew Law, Lisa Maher, Christopher K Fairley, Steven Skov, Nathan Ryder, Elizabeth Moore, Jacqueline Mein, Carole Reeve, Donna Ah Chee, John Boffa and John M Kaldo

Brain energy rescue:an emerging therapeutic concept for neurodegenerative disorders of ageing

The brain requires a continuous supply of energy in the form of ATP, most of which is produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. In neurodegenerative disorders of ageing, brain glucose metabolism deteriorates in a progressive, region-specific and disease-specific manner — a problem that is best characterized in Alzheimer disease, where it begins presymptomatically. This Review discusses the status and prospects of therapeutic strategies for countering neurodegenerative disorders of ageing by improving, preserving or rescuing brain energetics. The approaches described include restoring oxidative phosphorylation and glycolysis, increasing insulin sensitivity, correcting mitochondrial dysfunction, ketone-based interventions, acting via hormones that modulate cerebral energetics, RNA therapeutics and complementary multimodal lifestyle changes