REPORTS
measured in a larger population. The method of choice for measuring a defined, diagnostic panel of miRNAs is RT-qPCR. To perform RT-qPCR, RNA must be
extracted from plasma or serum and reverse transcribed. Target miRNAs must then be amplified in individual PCR reactions. This process takes several hours and is sufficient for the current applications for detecting circu- lating miRNA biomarkers for which results may be reported within days or weeks. However, a more rapid assay would facilitate point-of-care diagnostics and expand potential uses. For example, changes in miRNAs have been associated with cardiac disease (10), and the ability to measure them in plasma quickly might provide earlier biomarkers for diagnosis of myocardial infarction and ensure more timely thera- peutic intervention (13). We therefore assessed the ability
of the xxpress system (BJS Biotech- nologies, Perivale, UK) to quantify miRNAs in plasma using high speed cycling in comparison with an existing qPCR system (LightCycler 480; Roche, Basel, Switzerland). It was possible to amplify RNAs from plasma cDNA in less than 10 min using the xxpress system, compared with ~40 min with the Light- Cycler. However, although the perfor- mance of both systems was broadly comparable, variability across the plate and between replicate samples was greater in the xxpress.
Materials and methods
Oligonucleotide synthesis All oligonucleotides (Integrated DNA Technologies, Leuven, Belgium) were reconstituted as 100 µM stocks, DNA in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and RNA in nuclease free water, and then stored at -80°C. Sequences of the RNA miRNA mimic, reverse transcription oligo, 18S primers, and miRNA primers are shown in Table 1.
qPCR instruments The xxpress system (Figure 1) employs resistive heating and forced air cooling to facilitate rapid changes in temper- ature. PCR reactions are performed within proprietary “xxplates,” which comprise a metal base fused with plastic
Vol. 58 | No. 5 | 2015 A)
0 B)
5
10
15
20
25 cm
Figure 1. The xxpress system. (A) A 96-well xxplate (bottom) compared with conventional 96-well and 384-well PCR plates (upper). (Scale bar represents 25 cm to allow a direct size comparison.) (B) The xxpress system comprises a heat sealer (left), centrifuge (center), and cycler unit (right).
Table 1. Oligonucleotides. Name
Sequence (5´-3´)
RT oligos miR-21 miRNA mimic UAG CUU AUC AGA CUG AUG UUG AAA AAA AAA AAA A OligodT-RACE Reverse RACE 18S forward 18S reverse
18S
miRNAs miR-10b-5p miR-468–5p miR-126–5p miR-22–3p miR-21–5p
Y-RNAs Y-RNA-3p Y-RNA-5p
TM
(°C) 52.4
GCG AGC ACA GAA TTA ATA CGA CTC ACT ATA GGT TTT TTT TTT TTV N 61.9 GCG AGC ACA GAA TTA ATA CGA C AAA CGG CTA CCA CAT CCA AG CCT CCA ATG GAT CCT CGT TA TAC CCT GTA GAA CCG AAT TTG TG TCC TGT ACT GAG CTG CCC CGA CAT TAT TAC TTT TGG TAC GCG AAG CTG CCA GTT GAA GAA CTG T TAG CTT ATC AGA CTG ATG TTG A CCC CCA CTG CTA AAT TTG ACT GGC TGG TCC GAT GGT AGT
53.9 55.3 53.8 54.7 62.8 49.6 57.1 50.9 55.2 56.8
calculated by Integrated DNA Technologies. 245
Sequence and melting temperatures for oligonucleotides used in this investigation. Column 1 denotes the general class of oligonucleotides used, and Column 2 denotes the name of each, also providing information about target strand (5p or 3p for miRNAs). Column 3 provides sequence information (V denotes any nucleotide not T or U, and N denotes any nucleotide). Column 4 TM
values were
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