title: Rapid quantification of microRNAs in plasma using a fast real-time PCR system.
abstract: 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) amplification. 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 quantification cycle (Cq) for detection of 18S rDNA from a standard genomic DNA sample was significantly more variable across the block (F-test, P = 2.4 × 10(-25)) 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.
authors.affiliation: Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: William John
authors.initials: WJ
authors.lastname: Andrews
authors.affiliation: Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Eoin Daniel
authors.initials: ED
authors.lastname: Brown
authors.affiliation: Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Margaret
authors.initials: M
authors.lastname: Dellett
authors.affiliation: Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: Ruth Esther
authors.initials: RE
authors.lastname: Hogg
authors.affiliation: Centre for Experimental Medicine, Queen's University Belfast, Belfast, Northern Ireland, United Kingdom.
authors.forename: David Arthur
authors.initials: DA
authors.lastname: Simpson
mesh_terms: D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D035683:MicroRNAs
keywords: PCR; RT-PCR; biomarker; microRNA