REPORTS


both systems (Figure 3B). Given the high concentrations of specific template miRNA used in this assay, a better signal may be obtained using cell-derived RNA as template (15). Our ultimate aim is the rapid detection


of miRNAs from blood as biomarkers of cancer and other diseases. As a step toward this goal, we measured the expression of several miRNAs in RNA extracted from three human plasma samples: miR-21, an “oncomir” that plays a role in many types of cancer (16–18) and heart disease (19) and has been reported to be elevated in blood from cancer patients (20,21); miR-22, which is also differentially expressed in various types of cancer (22); miR-126, which is involved in regulation of angiogenesis (23); and miR-486, which is down-regulated in lung cancer and proposed as a biomarker for detection of lung cancer in plasma samples (24,25). The results of quanti- fication of these 4 miRNAs following 40 cycles of PCR, completed in less than 10 min with the xxpress compared with 40 min using the LightCycler, are presented in Figure 4. The relative abundances of the miRNAs detected by both systems


were similar, albeit with greater variation between technical replicates on the xxpress. MiR-21 was the most abundant (lowest Cq


) miRNA and miR-22 the least


abundant (highest Cq) for all samples on values were


both platforms. Significant differences between platforms in Cq


observed for many miRNAs amplified from the same sample (e.g., miR-126 in plasma sample 2; P < 0.001). This is likely due to the combined effects of differences in the sample temperatures achieved during cycling, the detection systems, and the performance of the individual assays under different condi- tions. Another class of small RNA, a Y RNA fragment (hY4–3p) recently shown to be present in plasma (26–28) was detected consistently by both platforms (∆Cq


between the 2 platforms across


3 plasma samples was 2.3 ± 0.3 SD) and could potentially provide a stable reference gene. Faster PCR can be achieved by


reducing hold times, and although this can reduce sensitivity and increase variability (29), enzymes are now available from a range of manufacturers that perform well in fast PCR. All data


reported were generated using SYBR fast qPCR Mastermix (Kapa Biosystems), but 18S rDNA failed to amplify using Quantitect (Qiagen) on the xxpress, so users should be aware that rigorous optimization of different mastermixes is required for fast PCR. To facilitate devel- opment of rapid assays incorporating fast PCR, pre-processing steps must be minimized. There is therefore a growing need to develop enzymes resistant to contaminants and inhibitors present in a range of biological sample materials, such as soil, water and blood (30). Although many prototype rapid


PCR systems have been reported with extremely fast ramp rates and minia- turization (5,6,31), very few proceed to commercial release. Therefore, the xxpress rapid thermal cycler, particu- larly given its standard block-based design, offers a unique opportunity for the wider molecular research community to adopt fast PCR. The potential applica- tions of fast PCR in situations that require a rapid diagnosis [e.g., involvement of cardiac disease in dyspnoea (32) or chest pain] make this an exciting and rapidly expanding area of PCR devel-


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