biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsLiwen Wutranscription profiling by RT-PCRHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-64915TaqMan low density array (TLDA) was carried out to screen of the profiles of circulating miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients. The expression changes of circulating miRNAs in osteosarcoma patients were identified. To select candidate plasma miRNAs for osteosarcoma detection and monitoring, we employed TLDA technique to screen expression levels of 739 miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients (each pooled from 10 individuals).biostudies-arrayexpressGrowth Protocol - n/aNucleic Acid Extraction - Total RNA was isolated from each pool of plasma samples using the mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturerM-bM-^@M-^Ys instructions.Labeling - For cDNA synthesis, 30M-bM-^@M-^Ing of total RNA was subjected to reverse transcription using a TaqManM-. microRNA Reverse Transcription Kit (#4366596; Applied Biosystems) and Megaplex RT primers (Human Pool A + B; Applied Biosystems) following the manufacturerM-bM-^@M-^Ys protocol, allowing simultaneous reverse transcription of 739 mature human miRNAs to generate a miRNA cDNA library corresponding to each plasma sample. Reverse transcription was performed with the following cycling conditions: 40M-bM-^@M-^Icycles at 16M-0C for 2M-bM-^@M-^Imin, 42M-0C for 1M-bM-^@M-^Imin and 50M-0C for 1M-bM-^@M-^Is followed by a final step of 80M-0C for 5M-bM-^@M-^Imin to inactivate reverse transcriptase. Thereafter, to generate enough miRNA cDNA template for the following real-time PCR, the cDNA libraries were pre-amplified using Megaplex PreAmp primer and PreAmp Master Mix (Applied Biosystems) following the manufacturerM-bM-^@M-^Ys instructions. The PreAmp primer pool used here consisted of forward primers specific for each of the 739 human miRNAs and a universal reverse primer. The pre-amplification cycling conditions were as follows: 95M-0C for 10M-bM-^@M-^Imin, 55M-0C for 2M-bM-^@M-^Imin, 72M-0C for 2M-bM-^@M-^Imin followed by 12M-bM-^@M-^Icycles at 95M-0C for 30M-bM-^@M-^Is and 60M-0C for 4M-bM-^@M-^Imin; the samples were then held at 99.9M-0C for 10M-bM-^@M-^Imin. After the preamplification step, the products were diluted with RNase-free water, combined with TaqMan gene expression Master Mix and then loaded into TaqMan TaqMan Low Density Array (TLDA) (Applied Biosystems), which are two 384-well formatted plates and real-time PCR-based microfluidic cards with embedded TaqMan primers and probes in each well for the 739 different mature human miRNAs. Real-time PCR was performed on an ABI PRISM 7900HT sequence detection system (Applied Biosystems) with the following cycling conditions: 50M-0C for 2M-bM-^@M-^Imin, 94.5M-0C for 10M-bM-^@M-^Imin followed by 40M-bM-^@M-^Icycles at 95M-0C for 30M-bM-^@M-^Is and 59.7M-0C for 1M-bM-^@M-^Imin.Sample Treatment - The pre-operative blood samples were obtained from 90 patients prior to surgery and/or chemotherapy. The control blood samples were obtained from healthy people. All extracted plasma samples were stored in phased liquid nitrogen. To minimize the effect of freeze-thaw on circulating miRNAs, we only used plasma samples which had not been previously thawed.Scaning - n/aHybridization - n/aMIAME ScoreOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsLiwen WuFeng LianData Transformation - The Ct (cycle threshold) was automatically given by SDS 2.3 software (Applied Biosystems) and is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. U6 embedded in the TaqMan Human MicroRNA Arrays was used as an internal control. The relative expression levels of miRNAs were calculated using the comparative M-NM-^TM-NM-^TCt method. ID_REF = VALUE = normalized signal (against housekeeping genes)falseIdentification of a plasma four-microRNA panel as a potential noninvasive biomarker for osteosarcomaTaqMan low density array (TLDA) was carried out to screen of the profiles of circulating miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients. The expression changes of circulating miRNAs in osteosarcoma patients were identified. To select candidate plasma miRNAs for osteosarcoma detection and monitoring, we employed TLDA technique to screen expression levels of 739 miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients (each pooled from 10 individuals).2015-01-13T00:00:00Z2023-10-13T14:03:54.029Z2022-02-02T15:32:27.941ZE-GEOD-64915GSE64915