Quantification of Circulating miRNAs by Droplet Digital PCR: Comparison of EvaGreen- and TaqMan-Based Chemistries

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 DNAbinding 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/mL. 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.

Paired Box 5 Methylation Detection by Droplet Digital PCR for Ultra-Sensitive Deep Surgical Margins Analysis of Head and Neck Squamous Cell Carcinoma

“…The QX200 droplet generator (Bio-rad Laboratories) created an average of 20,000 oil droplets per sample well and clouded droplet samples were transferred to a 96-well PCR plate (Eppendorf, Hauppauge, NY). The plate was heat-sealed with foil and placed in a thermal cycler under the following conditions: 95°C for 10 minutes, then 40 cycles of 95°C for 30 seconds and 60°C for 1 minute and two final steps at 98°C for 10 minutes and 4°C hold to enhance dye stabilization(22, 23). Finally, the plate was placed on the QX200 droplet reader (Bio-rad Labolatories).…”

Circulating tumor DNAs and non-coding RNAs as potential biomarkers for hepatocellular carcinoma diagnosis, prognosis and response to therapy

“…The most common technologies employed to measure miRNA expression in biological samples include microarray, NGS, quantitative real-time PCR (RT-qPCR) and droplet digital PCR (ddPCR) [36][37][38] . Microarray and NGS technologies are suitable for screening and discovery purposes, qPCR and ddPCR remain the choices for validation and clinical tests development.…”

“…Furthermore, ddPCR proved to be more tolerant than real time qPCR to the presence of inhibitors [39] . Finally, ddPCR generally exhibits a higher precision and reproducibility than real time qPCR, thus allowing an easier discrimination between cases and controls [37,40,41] .…”

Development of retroviral vectors for insertional mutagenesis in medaka haploid cells

“…Then ddPCR (Hindson et al, 2013;Miotto et al, 2014) was performed to quantify proviral DNA integrated in transduced cells, taking gfp as the target gene and β-acting as the reference gene. Briefly, ddPCR assay mixtures (25 μl) containing 12.5 μl 2× ddPCR™ EvaGreen Super Mix, 40-60 ng HindIII digested genomic DNA, and 100 nM primers (Supplementary Table 2S) were loaded onto the Automated Droplet Generator (Biorad) for droplet generation.…”