Ultra-precise quantification of mRNA targets across a broad dynamic range with nanoreactor beads

Abstract

Precise quantification of molecular targets in a biological sample across a wide dynamic range is a key requirement in many diagnostic procedures, such as monitoring response to therapy or detection of measurable residual disease. State of the art digital PCR assays provide for a dynamic range of four orders of magnitude. However digital assays are complex and require sophisticated microfluidic tools. Here we present an assay format that enables ultra-precise quantification of RNA targets in a single measurement across a dynamic range of more than seven orders of magnitude. The approach is based on hydrogel beads that provide for microfluidic free compartmentalization of the sample as they are used as digital nanoreactors for reverse transcription, PCR amplification and combined real time and digital detection of gene transcripts. We have applied these nanoreactors for establishing an assay for the detection and quantification of BCR-ABL1 fusion transcripts. The assay has been characterized for its accuracy and linear dynamic range. A comparison of the new method against the current clinical standard procedure with clinical samples from patients with chronic myeloid leukemia (CML) revealed good agreement between the results obtained with the respective method.