Open AccessPhysical and chemical template-blocking strategies in the exponential amplification reaction of circulating microRNAs
Author(s) -
Michael Patrick Trinh,
Jocelyn G. Carballo,
Gary Brent Adkins,
Kaizhu Guo,
Wenwan Zhong
Publication year - 2020
Publication title -
analytical and bioanalytical chemistry/analytical and bioanalytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.86
H-Index - 166
eISSN - 1618-2650
pISSN - 1618-2642
DOI - 10.1007/s00216-020-02496-w
Subject(s) - microrna , nucleic acid , template , chemistry , dna , loop mediated isothermal amplification , computational biology , biophysics , nanotechnology , combinatorial chemistry , microbiology and biotechnology , biology , materials science , biochemistry , gene
The detection of circulating miRNA through isothermal amplification wields many attractive advantages over traditional methods, such as reverse transcription RT-qPCR. However, it is challenging to control the background signal produced in the absence of target, which severely hampers applications of such methods for detecting low abundance targets in complex biological samples. In the present work, we employed both the cobalt oxyhydroxide (CoOOH) nanoflakes and the chemical modification of hexanediol to block non-specific template elongation in exponential amplification reaction (EXPAR). Adsorption by the CoOOH nanoflakes and the hexanediol modification at the 3' end effectively prevented no-target polymerization on the template itself and thus greatly improved the performance of EXPAR, detecting as low as 10 aM of several miRNA targets, including miR-16, miR-21, and miR-122, with the fluorescent DNA staining dye of SYBR Gold™. Little to no cross-reactivity was observed from the interfering strands present in 10-fold excess. Besides contributing to background reduction, the CoOOH nanoflakes strongly adsorbed nucleic acids and isolated them from a complex sample matrix, thus permitting successful detection of the target miRNA in the serum. We expect that simple but sensitive template-blocking EXPAR could be a valuable tool to help with the discovery and validation of miRNA markers in biospecimens. Graphical abstract.