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Supplementary Table 1 from Quantification of Circulating miRNAs by Droplet Digital PCR: Comparison of EvaGreen- and TaqMan-Based Chemistries
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posted on 2023-03-31, 14:07 authored by Elena Miotto, Elena Saccenti, Laura Lupini, Elisa Callegari, Massimo Negrini, Manuela FerracinIntra-run and inter-run variability of EvaGreen dye-based ddPCR assay for 10 samples. Results are presented as copies of cel-miR-39 per microliter of the amplification reaction. SD: standard deviation. CV coefficient of variation calculated over 3 replicates or over 3 days (overall precision).
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ARTICLE ABSTRACT
Droplet digital PCR (ddPCR) has been successfully used with TaqMan assays to assess gene expression through the quantification of mRNA and miRNA. Recently, a new ddPCR system that can also run DNA-binding dye-based assays has been developed but it has not yet been tested for miRNA. We tested and compared the feasibility of quantifying miRNA with the new QX200 Droplet Digital PCR system when used with EvaGreen dye– and TaqMan probe–based assays. RNA from plasma and serum of 28 patients with cancer and healthy persons was reverse-transcribed and quantified for two circulating miRNAs and one added exogenous miRNA, with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. Amplification and detection of target miRNAs were performed on the QX200 ddPCR system. Conditions required to run miRCURY LNA miRNA assays were optimized. The EvaGreen-based assay was precise, reproducible over a range of concentrations of four orders of magnitude, and sensitive, detecting a target miRNA at levels down to 1 copy/μL. When this assay was compared with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma (Pearson r > 0.90). EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs in human plasma and serum. This study establishes the basis for using EvaGreen dye–based assays on a ddPCR system for quantifying circulating miRNA biomarkers and potentially other low-abundance RNA biomarkers in human biofluids.See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.”Cancer Epidemiol Biomarkers Prev; 23(12); 2638–42. ©2014 AACR.Usage metrics
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