Reports Rapid quantification of microRNAs in plasma using


a fast real-time PCR system William John Andrews, Eoin Daniel Brown, Margaret Dellett, Ruth Esther Hogg, and David Arthur Simpson Centre for Experimental Medicine, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom


BioTechniques 58:244-252 (May 2015) doi 10.2144/000114287 Keywords: PCR; microRNA; RT-PCR; biomarker


Supplementary material for this article is available at www.BioTechniques.com/article/114287.


The ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers for a range of conditions. One rate-limiting factor in miRNA detection is the time taken to perform quantitative real-time PCR (qPCR) amplifica- tion. We therefore evaluated the ability of a novel thermal cycler to perform this step in less than 10 minutes. Quantitative PCR was performed on an xxpress thermal cycler (BJS Biotechnologies), which employs a resistive heating system and forced air cooling to achieve thermal ramp rates of 10°C/s, and a conventional Peltier-controlled LightCycler 480 system (Roche) ramping at 4.8°C/s. The quan- tification cycle (Cq


more variable across the block (F-test, P = 2.4 × 10-25


) for detection of 18S rDNA from a standard genomic DNA sample was significantly ) for the xxpress (20.01 ± 0.47 SD) than for the


LightCycler (19.87 ± 0.04 SD). RNA was extracted from human plasma, reverse transcribed, and a panel of miRNAs was amplified and detected using SYBR Green. The sensitivities of the two systems were broadly comparable—both detected a panel of miRNAs reliably, and both indicated similar relative abundances. The xxpress thermal cycler facilitates rapid qPCR detection of small RNAs and brings point-of-care diagnostics based upon detection of circulating miRNAs a step closer to reality.


PCR is ubiquitous throughout the life and medical sciences, and a reduction in the time required to complete a PCR reaction would therefore be of immense benefit. While the choice of a fast enzyme is important for the optimization of fast PCR-based systems (1,2), the speed at which the temperature of the sample can be altered during thermal cycling is the primary rate-limiting factor (3). Using prototype systems, many investigators have demonstrated that rapid thermal cycling is possible (4,5) and, under extreme conditions, can be completed in less than 1 min (6). The predominant format of existing thermal cyclers comprises a 96- or 384-well block, the temperature of which is controlled by a Peltier-based system


METHOD SUMMARY


Here we present a quantitative PCR platformthat enables faster ramping between temperatures than conventional Peltier-based systems, thereby reducing the time required to complete a PCR reaction. This is particularly important for the development of clinical biomarkers for acute conditions, and we demonstrate the feasibility of this approach for detecting miRNAs in plasma.


Vol. 58 | No. 5 | 2015 244 www.BioTechniques.com


limited to ramp rates of approximately 4°C/s. Several quantitative PCR systems that employ rapid thermal cycling are available commercially, but these are based on glass capillaries (LightCycler; Roche) (7) or plastic tubes placed in a centrifuge (Rotor-Gene; Qiagen). An alternative rapid plate-based approach, which can be more easily integrated into existing workflows, has now been developed. The xxpress thermal cycler (BJS Biotechnologies) employs resistive heating and forced air cooling to enable ramp rates of up to 10°C/s. One of the main applications of


PCR is for the quantitation of RNA targets. Following reverse transcription into cDNA, amplification of targets is detected by incorporation of a double-


stranded DNA binding fluorescent dye (principally SYBR Green) or use of a sequence-specific probe-based system (e.g., TaqMan). This quantitative reverse transcription PCR (RT-qPCR) approach can be modified to measure small RNAs, specifically microRNAs (miRNAs). Following the discovery that miRNAs exist in a stable form within blood (8,9), their potential as biomarkers was soon realized. miRNA expression patterns characteristic of cancer, cardiovas- cular disease, diabetes, Alzheimer’s, and many other conditions have now been reported (10–12). Typically, global miRNA profiles are initially assessed in a discovery cohort using microarrays or deep sequencing, and selected informative miRNAs are subsequently


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