REPORTS A)
15 17 19 21 23 25 27 29 31 33 35
xxpress LightCycler
LC480 xxpress
R² = 0.9547 R² = 0.9878
B)
10 12 14 16 18 20 22 24
0.00001 0.0001 0.001 0.01 0.1 Genomic DNA Template (ng/μl) 1 10 100
xxpress LightCycler
R² = 0.9506 R² = 0.9867
1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 1.E+10 RNA Copy Number
Figure 3. Standard curves generated by amplification of 18S rDNA from genomic DNA and synthetic miR-21. (A) 18S rDNA standard curve analysis. Com- parable dynamic range 0.1 pg–10 ng genomic DNA; amplified using the maximal ramp rate on each platform (n = 3 technical replicates) (*P < 0.05, **P < 0.01). (B) Synthetic miR-21 standard curve analysis. Comparable dynamic range: 109
–106 copies of template RNA; amplified using the maximal ramp rate on each platform (n = 3 technical replicates). For both data sets, melting curve analysis confirmed the presence of a single product (data not shown).
denatured at 50°C for 5 min, then cooled to 4°C for addition of Super- Script III (Life Technologies, Paisley, UK) followed by incubation at 40°C for 60 min. Total RNA, including small RNAs, was extracted from 200 µL human plasma using a miRNeasy serum/plasma kit according to the manufacturer’s protocol (Qiagen, Crawley, UK) and polyadenylated for 1 h at 37°C in a 25 µL reaction containing 2 U poly(A) polymerase, 2.5 mM MgCl2
,
and 1 mM ATP (Ambion, Austin, TX). A 5 µL volume of polyadenylated RNA was then incubated at 65°C for 5 min with 0.5 µg oligo(dT)-RACE primer and 1 µl 10 mM dNTPs in a reaction volume of 8 µL. Following addition of 200 U SuperScript III, 1 U RNaseOut, 1 µL 0.1 M DTT, and 4 µL 5× RT buffer, the reaction was incubated at 50°C for 1 h followed by 70°C for 15 min.
Ethics This study was conducted according to the guidelines laid down in the Decla- ration of Helsinki, and all procedures involving human participants/patients were approved by the Research Ethics Committee of the School of Medicine and Dentistry, Queen’s University Belfast (Ref:11/05v3). Written informed consent was obtained from all participants.
Vol. 58 | No. 5 | 2015 Results and discussion
To assess consistency across the plate, a single mastermix for amplification of a 155 bp amplicon from 18S rDNA was aliquoted into 48 wells distributed evenly across each type of plate (Supplementary Figure S1). Amplification was performed using the same cycling conditions on
both systems (Table 2). The mean Cq values were very similar for the xxpress (20.01 ± 0.47 SD) and the LightCycler (19.87 ± 0.04 SD), although the Light- Cycler was significantly lower (t-test, P = 0.035). However, the xxpress exhibited significantly greater variability than the LightCycler, which generated extremely consistent Cq
values (F-test, P = 2.4 × 10-25 ) (Figure 2). Although the perfor-
mance of the xxpress was less robust, it could still be used in situations requiring rapid detection of large variations in expression between samples (with the inclusion of appropriate replicates). The spread in Cq
values on the xxpress
was in part linked to the position of the sample on the xxplate, with adjacent
wells tending to vary from the mean Cq value in the same direction. The greater variation observed with the xxpress may not be due to poorer temperature control across the plate, but could be related to the optical system, which is amenable to
248
improvement in subsequent versions. To compare the sensitivity of the two platforms, highly abundant 18S rDNA was amplified from a dilution series of genomic DNA. The mean Cq
values at
each template concentration were not significantly different between platforms (t-test, P > 0.05). In both cases, the Cq
template concentration (LightCycler, R2 = 0.9547; xxpress, R2
= 0.9878), although
again there was a significantly greater variation in Cq
values for replicates in
the xxpress at several concentrations (Figure 3A). The method described by Shi and
Chiang (14) was then used to detect miRNAs. Total RNA, including miRNAs, is polyadenylated and then reverse- transcribed with a poly(T) adapter into cDNAs. These are subsequently amplified using an miRNA-specific forward primer and a sequence complementary to the poly(T) adapter as the reverse primer. Initially, we tested the ability of each platform to detect an miRNA mimic synthesized with a poly(A) tail already incorporated (thereby removing any variability associated with the polyade- nylation reaction). The limit of detection, as estimated from the linear portion of the standard curve, was approximately 1 × 106
values were highly correlated with
RNA template molecules for
www.BioTechniques.com
Cq
Cq
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