Measuring microRNAs: Comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods

<p>Abstract</p> <p>Background</p> <p>Determining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be affected by the particular method of total RNA preparation used.</p> <p>Results</p> <p>We measured the expression of 470 human miRNAs in nine human tissues using Agilent microarrays, and compared these results to qPCR profiles of 61 miRNAs in the same tissues. Most expressed miRNAs (53/60) correlated well (R > 0.9) between the two methods. Using spiked-in synthetic miRNAs, we further examined the two miRNAs with the lowest correlations, and found the differences cannot be attributed to differential sensitivity of the two methods. We also tested three widely-used total RNA sample prep methods using miRNA microarrays. We found that while almost all miRNA levels correspond between the three methods, there were a few miRNAs whose levels consistently differed between the different prep techniques when measured by microarray analysis. These differences were corroborated by qPCR measurements.</p> <p>Conclusion</p> <p>The correlations between Agilent miRNA microarray results and qPCR results are generally excellent, as are the correlations between different total RNA prep methods. However, there are a few miRNAs whose levels do not correlate between the microarray and qPCR measurements, or between different sample prep methods. Researchers should therefore take care when comparing results obtained using different analysis or sample preparation methods.</p

ANALISIS PENENTUAN LOKASI PEMBANGUNAN PABRIK PENGOLAHAN UDANG VANNAMEI DI KABUPATEN SUMENEP

Kabupaten Sumenep memiliki potensi budidaya tambak udang Vannamei yang sangat besar, potensi tersebut dapat dioptimalkan dengan meningkatkan nilai tambah dari udang Vannamei. Salah satu langkah untuk mengoptimalkan potensi besar tersebut adalah dengan mengolah hasil produksi dari tambak udang Vannamei, yang masih berupa bahan mentah menjadi produk olahan yang memiliki nilai ekonomi yang tinggi. Penelitian ini bertujuan untuk menentukan lokasi yang paling sesuai untuk digunakan sebagai lokasi pabrik pengolahan udang Vannamei di Kabupaten Sumenep. Jenis Penelitian yang digunakan adalah penelitian deskriptif kualitatif, dengan menggunakan metode analisis kontinuitas bahan baku, AHP (Analitycal Hierarchy Process) dan analisis Overlay. Hasil dari penelitian ini menunjukkan bahwa lokasi yang paling sesuai untuk digunakan sebagai lokasi pabrik pengolahan udang Vannamei di Kabupaten Sumenep terletak pada Kecamatan Dungkek, Batang-Batang dan Gapura, dengan didukung tingkat produksi bahan baku tinggi, memiliki infrastruktur yang baik, serta sesuai dengan arahan rencana tata ruang wilayah Kabupaten Sumenep. Kata Kunci: Penentuan Lokasi, Pabrik Pengolahan, Udang Vanname

In Vitro Evaluation of Oxoplatin: An Oral Platinum(IV) Anticancer Agent

Platinum(IV) compounds like oxoplatin (cis, cis, trans-diammine-dichlorido-dihydroxido-platinum(IV)) show increased stability and therefore can be applied orally. In a panel of 38 human cancer cell lines this drug induced S-phase arrest and cell death with IC50 values 2.5-fold higher than cisplatin. Oxoplatin may be converted to cisplatin by intracellular reducing agents, however, exposure to 0.1 M HCl mimicking gastric acid yielded cis-diammine-tetrachlorido-platinum(IV) exhibiting twofold increased activity. Similar results were obtained for another platinum(IV) compound, JM 149 (ammine-dichlorido-(cyclohexylamine)-dihydroxido-platinum(IV)), but not for its parent drug JM 216/satraplatin. Genome-wide expression profiling of H526 small cell lung cancer cells treated with these platinum species revealed clear differences in the expression pattern of affected genes between oxoplatin and cisplatin. In conclusion, oxoplatin constitutes a potent oral agent that is either reduced or converted to distinct active compounds, for example, by gastric acid or acidic areas prevailing in solid tumors, in dependence of the respective pharmaceutical formulation

Robust interlaboratory reproducibility of a gene expression signature measurement consistent with the needs of a new generation of diagnostic tools

The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. While the reproducibility of microarray gene expression measurements has been the subject of several recent reports, there is still a need for systematic investigation into what factors most contribute to variability of measured expression levels observed among different laboratories and different experimenters.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Spectrally resolved autofluorescence imaging in posterior uveitis.

Clinical discrimination of posterior uveitis entities remains a challenge. This exploratory, cross-sectional study investigated the green (GEFC) and red emission fluorescent components (REFC) of retinal and choroidal lesions in posterior uveitis to facilitate discrimination of the different entities. Eyes were imaged by color fundus photography, spectrally resolved fundus autofluorescence (Color-FAF) and optical coherence tomography. Retinal/choroidal lesions' intensities of GEFC (500-560 nm) and REFC (560-700 nm) were determined, and intensity-normalized Color-FAF images were compared for birdshot chorioretinopathy, ocular sarcoidosis, acute posterior multifocal placoid pigment epitheliopathy (APMPPE), and punctate inner choroidopathy (PIC). Multivariable regression analyses were performed to reveal possible confounders. 76 eyes of 45 patients were included with a total of 845 lesions. Mean GEFC/REFC ratios were 0.82 ± 0.10, 0.92 ± 0.11, 0.86 ± 0.10, and 1.09 ± 0.19 for birdshot chorioretinopathy, sarcoidosis, APMPPE, and PIC lesions, respectively, and were significantly different in repeated measures ANOVA (p < 0.0001). Non-pigmented retinal/choroidal lesions, macular neovascularizations, and fundus areas of choroidal thinning featured predominantly GEFC, and pigmented retinal lesions predominantly REFC. Color-FAF imaging revealed involvement of both, short- and long-wavelength emission fluorophores in posterior uveitis. The GEFC/REFC ratio of retinal and choroidal lesions was significantly different between distinct subgroups. Hence, this novel imaging biomarker could aid diagnosis and differentiation of posterior uveitis entities

Retest variability and patient reliability indices of quantitative fundus autofluorescence in age-related macular degeneration: a MACUSTAR study report

This study aimed to determine the retest variability of quantitative fundus autofluorescence (QAF) in patients with and without age-related macular degeneration (AMD) and evaluate the predictive value of patient reliability indices on retest reliability. A total of 132 eyes from 68 patients were examined, including healthy individuals and those with various stages of AMD. Duplicate QAF imaging was conducted at baseline and 2 weeks later across six study sites. Intraclass correlation (ICC) analysis was used to evaluate the consistency of imaging, and mean opinion scores (MOS) of image quality were generated by two researchers. The contribution of MOS and other factors to retest variation was assessed using mixed-effect linear models. Additionally, a Random Forest Regressor was trained to evaluate the extent to which manual image grading of image quality could be replaced by automated assessment (inferred MOS). The results showed that ICC values were high for all QAF images, with slightly lower values in AMD-affected eyes. The average inter-day ICC was found to be 0.77 for QAF segments within the QAF8 ring and 0.74 for peripheral segments. Image quality was predicted with a mean absolute error of 0.27 on a 5-point scale, and of all evaluated reliability indices, MOS/inferred MOS proved most important. The findings suggest that QAF allows for reliable testing of autofluorescence levels at the posterior pole in patients with AMD in a multicenter, multioperator setting. Patient reliability indices could serve as eligibility criteria for clinical trials, helping identify patients with adequate retest reliability

lincRNAs act in the circuitry controlling pluripotency and differentiation

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.Broad InstituteHarvard UniversityNational Human Genome Research Institute (U.S.)Merkin Family Foundation for Stem Cell Researc