Dye-based High-Throughput qPCR in Microfluidic Platform BioMark

Svec, D., Rusnakova, V., Korenkova, V., Kubista, M. Dye-based High-Throughput qPCR in Microfluidic Platform BioMark. In PCR Technology, Nolan, T. (ed.). CRC Press Inc., 323-339, 2013. ISBN 9781439848050.

Introduction

In biological and medical studies, large-scale gene expression and transcriptome analyses are essential tools.1 Based on the goals of the investigator, different methods such as RNA microarrays, massive parallel sequencing, or high-throughput real-time quantitative PCR (qPCR) can be used. There are currently four platforms available for high-throughput qPCR: the BioMark from Fluidigm, the OpenArray from Life Technologies, the LightCycler™ 1536 from Roche, and the Smartchip™ from Wafergen (Table 23.1). These instruments offer the broadest dynamic range, high versatility of experiment setup, low cost per sample, and the lowest demands on the amount of sample material, allowing analysis of minute sample amounts, including single cells.2,3 One focus of our center is gene expression profiling of single cells and we have used the BioMark as well as the OpenArray system. When we acquired the BioMark, most of our assay libraries were based on nonspecific dyes and so we explored using these established dye-based assays in microfluidic qPCR and extensively optimized the experimental protocol. In this report, we focus on the use of the BioMark system, which uses microfluidic chips called “dynamic arrays” for gene expression profiling, “digital arrays” for digital PCR, and reusable genotyping arrays for single nucleotide polymorphism (SNP) applications.....