Profiling of circulating microRNAs in patients with Barrettâ??s esophagus and esophageal adenocarcinoma
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ABSTRACT: miRNAs were extracted from plasma samples of healthy volunteers, patients with Barrettâ??s esophagus and patients with esophageal adenocarcinoma. We used the Serum / Plasma Focus miRNA PCR panel (Exiqon, Denmark) to quantitate miRNA presence in the different patient groups. PCR miRNA profiling. miRNA from three different patient groups were extracted from their plasma.
Project description:Plasma samples from 10 colonrectal cancer patients (CRCs) (including 5 stage II and 5 stage III patients) and 10 normal controls.An amount of 5 to 10 milliliters of whole blood were obtained from each participant.The plasma was obtained by centrifugation at 1200g for 10min at 4°C.To complete the removal of residual cellular components, plasma samples were recentrifuged at 12,000g for a further 10min at 4°C.A volume of 600?L of each plasma samples from CRC group or normal control group was picked out and uniformly mixed. qPCR miRNA expression profiling. Plasma samples form 10 colonrectal cancer patients(CRCs) (including 5 stage II and 5 stage III patients) and 10 normal controls were used and treated as indicated in the summary. Equal amount total RNA from each donor was pooled prior to gene expression analysis.
Project description:RNA was isolated with the RNeasy kit (Qiagen) from electronically sorted total CD4SP thymocytes. RNA was reverse transcribed into cDNA by oligo(dT) priming with the QuantiTect Reverse transcription kit (Qiagen). The expression of 84 key genes involved in apoptosis, or programmed cell death was performed with mouse apoptosis RT2ProfilerTM PCR array system (PAMM-012 E; SABiosciences). PCR Array data were analyzed by the Ct data analysis method. qPCR gene expression profiling. CD4SP thymocytes from WT and Ik7Tg mice were used.
Project description:RNA was isolated with the RNeasy kit (Qiagen) from electronically sorted immature CD4SP thymocytes. RNA was reverse transcribed into cDNA by oligo(dT) priming with the QuantiTect Reverse transcription kit (Qiagen). The expression of 84 key genes involved in apoptosis, or programmed cell death was performed with mouse apoptosis RT2ProfilerTM PCR array system (PAMM-012 E; SABiosciences). PCR Array data were analyzed by the Ct data analysis method. qPCR gene expression profiling. CD4SP thymocytes from WT and Ik7Tg mice were used.
Project description:RNA was isolated with the RNeasy kit (Qiagen) from electronically sorted mature CD4SP thymocytes. RNA was reverse transcribed into cDNA by oligo(dT) priming with the QuantiTect Reverse transcription kit (Qiagen). The expression of 84 key genes involved in apoptosis, or programmed cell death was performed with mouse apoptosis RT2ProfilerTM PCR array system (PAMM-012 E; SABiosciences). PCR Array data were analyzed by the Ct data analysis method. qPCR gene expression profiling. CD4SP thymocytes from WT and Ik7Tg mice were used.
Project description:Murine macrophages were isolated from the lungs of mice given a pulmonary challenge with C. neoformans strain H99. Mice were either given a protective (H99γ) or a mock (HKCn) immunization prior to C. neoformans H99 challenge, and macrophages were isolated from the lungs of mice 24 hours, 3 days, or 7 days post-challenge using anti-CD11b microbeads according to the Miltenyi cell sorting system. We used SA Biosciences Toll-like Receptor PCR assay panel to quantitate gene expression of signal transduction factors in total RNA isolated from macrophages derived from immunized mice compared to non-immunized. qPCR gene expression profiling. Macrophages from 5 mice per group were pooled and assayed as indicated in the summary. Each experiment was performed 3 times and the resulting Ct values of each group from each experiment averaged prior to data analysis. TIme points were analyzed separately
Project description:MCF10AT1 (M-II), MCF10CA1h (M-III) and MCF10CA1a.cl1 (M-IV) are a set of human breast cancer cell lines that derived from the same parent cell line MCF10 but display distinct phenotypes in metastatic potential in the form of xenografts in immune-compromised mice. From M-II to M-IV, the metastatic potential is gradually increased. Using a self-designed real-time PCR based miRNA array containing 242 genes, we tested the miRNA expression levels in these cell lines in order to identify metastasis-related miRNAs. qPCR gene expression profiling. Equal amount total RNA from each cell line was pooled prior to gene expression analysis.
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Normal and scleroderma fibroblasts were serum-starved for 24 hours and incubated in the presence or absence of TGF-β1 (2ng/ml) for 6 hours. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
Project description:Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level are known to take part in the control of bone formation and bone resorption. Recently, targeted secretion of miRNAs from cells originating from various tissues has been described, which allows for their minimal-invasive detection in serum/plasma and use as biomarkers for presence and progression of pathological conditions. One pilot study has reported circulating miRNAs in serum and tissue of fracture patients. However, further studies are required to explore whether a dysbalance in bone homeostasis of fracture patients can reliably be reflected by specific circulating miRNAs, and whether these miRNAs might serve as drugable targets. Here, we report results from a comprehensive multiplex study of 175 miRNAs in serum samples obtained from 7 patients with osteoporotic fractures at the femoral neck, and 7 age-matched controls. Following elaborate quality control statistical analysis of this exploratory dataset identified 9 microRNAs with altered serum levels in response to fracture (adjusted p-value < 0.1). Of these, hsa-miR-10a/b gave excellent discrimination of both groups (AUC = 1.0), and clustering of samples based on the top10 miRNAs confirmed the high discriminatory power of circulating microRNAs for osteoporotic fractures. In the next step 3 miRNAs with unknown roles in osteogenic differentiation and 4 miRNA from a previous study were tested for their effects on osteogenic differentiation. Of these, 3 miRNAs showed robust effects on osteogenic differentiation. Overall, these data provide important insights into changes in serum miRNA in post-traumatic patients. Future studies will show, whether this knowledge can be used to improve current diagnostic methodologies to predict fracture risk and design novel treatment strategies for osteoporosis patients. Two groups with n=7 per group; one groups represents cases with osteoporotic fractures, the control group is age-matched without fractures
Project description:MicroRNAs (miRNAs) are aberrant expressed in hepatocellular carcinoma (HCC) tissue and play a central role in diverse biological processes. We conducted a genome-wide miRNAs screening in 10 pairs of HCC tumor and adjacent non-tumor tissues to test the hypothesis that dysregulation of miRNAs in HCC tumor tissue are partially due to aberrant methylation in relevant miRNAs host genes. Taqman low density arrays were used to examine miRNA profiles in paired HCC tissues, and quantitative RT-PCR was used to validate candidate miRNAs for both discovery and validation sets. A cross-sectional study was conducted in 10 HCC tumor tissues and 10 adjacent non-tumor tissues in Columbia University Medical Center (CUMC), which is approved by the Institutional Review Board.
Project description:Jeko-1 cells were culture with or without PRC2 inhibitor 3-deazaneplanocin A (DZNep)(1uM) for 72hrs. We used TaqManM-BM-. Human MicroRNA Array to quantitate miRNA expression profile regulated by PRC2. qPCR miRNA expression profiling.