Digital amplification techniques offer the advantage of absolute quantification with exquisite precision.
Current digital assay technologies are complex and require sophisticated microfluidic tools, which inhibit their uptake and implementation. We have developed a new technique for generating digital compartments using a new class of molecular reagents – BLINK Beads. These hyrdrogel beads serve as volume templates for aqueous partitions of the sample, allowing for micro-fluidics free digital assays.
We have developed a new technique for generating digital compartments using a new class of molecular reagents – BLINK Beads.
Here we present an assay format that enables ultra-precise quantification of RNA targets in a single measurement across a measurement range of more than seven orders of magnitude.The assay set-up provides a detection format that combines endpoint (digital) and real-time detection in each individual nanoreactor compartment.
Using the BLINK Bead nanoreactors we created an assay for the detection and quantification of the amount of BCR-ABL mRNA relative to the internal reference gene transcript GUSB and characterized the assay for its precision and measurement range. We also performed a method comparison against the current clinical standard.
Chronic myeloid leukemia (CML) is characterised by the presence of the BCR-ABL fusion transcript. Quantification of the BCR-ABL transcript level reflects leukemic burden. Molecular response to treatment is determined based on the ratio of BCR-ABL transcript levels and a control gene.
In this paper we present the results of the evaluation of the new assay and compare the data with the current clinical gold standard. The quantitative results showed good agreement with the laboratory gold standard. Utilising nanoreactor beads, we created an assay with a measurement range of >6 logs. This design approach provides for high sensitivity and an unprecedented measurement range in a single reaction. Moreover, the assay is simple to perform and delivers quantitative results comparable with the current laboratory standard.
Read the paper: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242529