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

PLoS One. 2021 Mar 18;16(3):e0242529. doi: 10.1371/journal.pone.0242529. eCollection 2021.

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 six orders of magnitude. The approach is based on hydrogel beads that provide for microfluidic free compartmentalization of the sample as they are used as nanoreactors for reverse transcription, PCR amplification and combined real time and digital detection of gene transcripts. We have applied these nanoreactor beads for establishing an assay for the detection and quantification of BCR-ABL1 fusion transcripts. The assay has been characterized for its precision and linear dynamic range. A comparison of the new method against conventional real time RT-PCR analysis (reference method) with clinical samples from patients with chronic myeloid leukemia (CML) revealed excellent concordance with Pearsons correlation coefficient of 0.983 and slope of 1.08.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • DNA Primers / metabolism
  • Fusion Proteins, bcr-abl / genetics*
  • Humans
  • Hydrogels / chemistry
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive / genetics*
  • Leukemia, Myelogenous, Chronic, BCR-ABL Positive / pathology
  • Nanotechnology
  • RNA, Messenger / analysis
  • RNA, Messenger / metabolism*
  • Real-Time Polymerase Chain Reaction / instrumentation
  • Real-Time Polymerase Chain Reaction / methods*

Substances

  • BCR-ABL1 fusion protein, human
  • DNA Primers
  • Hydrogels
  • RNA, Messenger
  • Fusion Proteins, bcr-abl

Grant support

The authors' affiliation BLINK AG received funding for this study in the form of grants from The German Federal Ministry of Education and Research (BMBF) (Grant No. 031B0526A) and from Free State of Thuringia and European Regional Development Fund (Grant No. EU 2017 FE 9005). BLINK AG used these grants to provide support in the form of salaries to TE, EE, SH, IL, ST, and SK. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.