Prognostic Molecular Signatures for Metastatic Potential in Clinically Low-Risk Stage I and II Clear Cell Renal Cell Carcinomas
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ABSTRACT: Presented is the differential expression of mRNA and miRNA in 44 Stage I and Stage II patients who developed metastasis within 5 years of nephrectomy, compared to 21 patients who remained disease free. Extracted RNA from nephrectomy specimens preserved in FFPE blocks was sequenced using RNAseq. MiRNA expression was done using the TaqMan OpenArray qPCR protocol.
Project description:Presented is the differential expression of mRNA and miRNA in 44 Stage I and Stage II patients who developed metastasis within 5 years of nephrectomy, compared to 21 patients who remained disease free. Extracted RNA from nephrectomy specimens preserved in FFPE blocks was sequenced using RNAseq. MiRNA expression was done using the TaqMan OpenArray qPCR protocol.
Project description:Murine lung miRNAs were profiled by rodent TaqMan OpenArray after flaxseed feeding and/or radiation exposure Lung tissue was obtained 48 hours after radiation or no exposure from three animals each: flaxseed/no radiation, flaxseed/radiation, control diet/no radiation, control diet/radiation. miRNA profiles were determined by OpenArray.
Project description:Total RNA was extracted from 400 ul of serum with the miRNeasy Serum/Plasma advanced Kit (Qiagen) and quantified using the Qubit microRNA assay kit (Thermo Fisher Scientific). cDNA templates were prepared using the TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific), starting from 10 ng of RNA. RT-qPCR carried out on a QuantStudio 12K Flex (Applied Biosystems) using the TaqMan OpenArray miRNA panel.
Project description:miRNA was extracted from a range of Oesophageal Adenocarcinoma cell lines using the miRNeasy Mini Kit (Qiagen, #217004, Chadstone, Australia) and RNase-free DNase Set (Qiagen, #79254, Chadstone, Australia). miRNA was reverse transcribed using a Custom OpenArray® miRNA RT pool (Life Technologies cat # A25630) and the TaqMan® microRNA Reverse Transcription Kit (Life Technologies cat # 4366596). cDNA Pre-amplifications were carried out with a Custom OpenArray® PreAmp pool (Life Technologies cat # 4485255) and TaqMan PreAmp Master Mix (Life Technologies cat # 4488593). PCR runs were performed using a QuantStudio™ 12K Flex Real-Time PCR System. Baseline qPCR expression profiling of miRNAs from 8 oesophageal Adenocarcinoma cell lines, prior to treating the cell lines with either 2Gy ionising radiation, 20 µM Cisplatin, or 50 µM 5-fluorouracil (5-FU).
Project description:Array Manufacturer: Applied Biosystems, Catalogue number: 4470187, Distribution: virtual, Technology: RT-PCR, PCR assay Each TaqMan® OpenArray® Human MicroRNA Panel, QuantStudio™ 12K Flex contains 754 well-characterized human miRNA sequences from the Sanger miRBase v14. All 754 assays have been functionally validated with miRNA artificial templates., Studies using this array
Project description:We report a kidney cancer tissue-based prognostic biomarker encompassing 15 genes (15G score) to classify patients into low versus high risk for recurrence after curative nephrectomy. The 15G score was independently associated with disease free survival adjusting for clinicopathologic variables as well as existing clinical risk calculators or nomograms. By improving risk stratification of patients with ccRCC, the 15G score has the capacity to facilitate selection of biopsy confirmed small renal cancers (T1a) for treatment versus surveillance; inform intensity and duration of surveillance after curative nephrectomy; and to potentially facilitate patient selection for adjuvant systemic therapy. We retrospectively identified 110 patients who underwent radical nephrectomy for ccRCC (discovery cohort). Patients who recurred were matched based on grade/stage to patients without recurrence. Capture whole transcriptome sequencing was performed on RNA isolated from archival tissue using the Illumina platform. We developed a gene-expression signature to predict recurrence/disease-free survival (DFS) using a 15-fold lasso and elastic-net regularized linear Cox model. We derived the 31-gene cell cycle progression (mxCCP) score using RNAseq data for each patient. Kaplan-Meier (KM) curves and multivariable Cox proportional hazard testing were used to validate the independent prognostic impact of the gene-expression signature on DFS, disease specific survival (DSS) and overall survival (OS) in two validation datasets (combined n=761).
Project description:Exosomes were purified from 250 ul matched plasma and serum samples using ExoQuickTm. The presence of particles consistent in size with exosomes (60-150nm) was confirmed using a Nanosight LM10. miRNA was extracted from exosomes using an miRNeasy Serum/Plasma kit (Qiagen, #217184). miRNA was reversed transcribed using a TaqMan® microRNA Reverse Transcription Kit (Life technologies, #4366596). miRNA profiling was performed with a high throughput TaqMan® OpenArray® Human microRNA panel (Life technologies, #4461104). The panel consisted of probes for 754 human miRNAs that are based on miRNA sequences derived from Sanger miRBase v14. MegaplexTM Primer Human Pool A v2.1 and Human Pool B v2.0 or v3.0