Quantification of mRNA Expression Using Single-Molecule Nanopore Sensing

Abstract

RNA quantification methods are broadly used in life science research and in clinical diagnostics. Currently, real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most common analytical tool for RNA quantification. However, in cases of rare transcripts or inhibiting contaminants in the sample, an extensive amplification could bias the copy number estimation, leading to quantification errors and false diagnosis. Single-molecule techniques may bypass amplification but commonly rely on fluorescence detection and probe hybridization, which introduces noise and limits multiplexing. Here, we introduce reverse transcription quantitative nanopore sensing (RT-qNP), an RNA quantification method that involves synthesis and single-molecule detection of gene-specific cDNAs without the need for purification or amplification. RT-qNP allows us to accurately quantify the relative expression of metastasis-associated genes MACC1 and S100A4 in nonmetastasizing and metastasizing human cell lines, even at levels for which RT-qPCR quantification produces uncertain results. We further demonstrate the versatility of the method by adapting it to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA against a human reference gene. This internal reference circumvents the need for producing a calibration curve for each measurement, an imminent requirement in RT-qPCR experiments. In summary, we describe a general method to process complicated biological samples with minimal losses, adequate for direct nanopore sensing. Thus, harnessing the sensitivity of label-free single-molecule counting, RT-qNP can potentially detect minute expression levels of RNA biomarkers or viral infection in the early stages of disease and provide accurate amplification-free quantification.

RNA Pore Translocation with Static and Periodic Forces: Effect of Secondary and Tertiary Elements on Process Activation and Duration

“…Nanopore translocation is a powerful single-molecule probing technique that has been used in diverse contexts: from studying the physical response of homopolymers [19-21, 32, 34, 39], to the topological friction in chains with knots and links [33,[40][41][42][43][44][45][46][47][48], for sequencing and analyzing biopolymers' secondary and tertiary structures [7,11,29,41,[49][50][51][52][53][54][55][56][57], and study RNA too [1][2][3][4][6][7][8].…”

RNA Pore Translocation with Static and Periodic Forces: Effect of Secondary and Tertiary Elements on Process Activation and Duration

“…Pore translation has recently allowed for distinguishing different types of tRNAs 1 and quantifying mRNA expression. 2 Measurements of ionic current blockades in nanopores 3 have been used to sequence RNAs 4 to probe salient features of their folding pathway 5 and to detect modified nucleobases. 6 Molecular dynamics simulations have shown that driving RNAs through pores of appropriate width can relay information about their compliance to structural deformations 7 and directional mechanical resistance.…”

In situsolid-state nanopore fabrication

“…Given the breakdown voltage depends approximately linearly on the membrane thickness, 143 this will result in nanopores forming at the thinnest part of the membrane. This technique has been demonstrated using a He + FIB 164 and EBL followed by RIE 12,158 to locally thin regions of the membrane. However, the large capital cost of these instruments limits the availability of this method for many researchers.…”

“…being developed for commercial applications such as DNA sequencing, [6][7][8] protein analysis, 9,10 and analyte detection in ultra-dilute samples. [11][12][13] As such, single-molecule biosensing devices hold promise for future applications such as early stage diagnostics and personalised medicine.…”

“…Interest in nanopore sensors has often focused on DNA sequencing, 19 however, these devices have been used for a wide range of applications. In particular, nanopores have been developed for protein fingerprinting, [20][21][22] polymer data storage, 23,24 biomarker detection, 12,25 enzymology, 26 ultrasensitive ion detectors, 27 nanoparticle fabrication, 28 and nanoscale chemical reactors 29,30 to name only some. Given this wide range of applications, it is important to be able to vary the nanopore dimensions and operate these devices in a wide range of environmental conditions.…”