Figure 1.
Comparison of cfDNA recovery using different commercially available kits.
A. Average cfDNA yield per 1 ml of plasma with 4 extraction kits, quantified using qPCR for GAPDH, ACTBL2 and HPRT and Qubit® 2.0 High Sensitivity dsDNA Assay. Error bar = ±SEM, n = 9 from 3 independent experiments each performed in triplicate. ND = Not detectable. B. Percent recovery of serial dilutions of λ/HindIII DNA, quantified using assays targeting the 23 kb and 564 bp fragments. Error bar = ±SEM, n = 9 from 3 independent experiments performed in triplicate. CNA = Circulating Nucleic Acid kit.
Figure 2.
Comparison of number of reads between two different extraction kits using the Ion AmpliSeq™Cancer Hotspot Panel v2.
Analysis of 4 plasma samples extracted using both the QIAamp® DNA Blood Mini kit and the QIAamp® CNA Kit. In each trace, the number of reads is shown on the Y-axis. Red lines indicate the QIAamp® CNA Kit and the blue lines represent the QIAamp® DNA Blood Mini kit. For in depth analysis see Tables S1 and S2.
Figure 3.
Bioanalyzer analysis of circulating miRNA iolated with different kits.
A-C. Representative Bioanalyser traces of cell-free RNA obtained from 1 ml of plasma using 3 commercial kits. The kits used and RNA integrity (RIN) values are highlighted above the trace and representative gels are shown below each trace. A = extractions using the miRNeasy Serum/Plasma kit, B = mirVana microRNA Isolation kit and C = Circulating Nucleic Acid kit. Quantities obtained are shown in D. Error bar = ±SEM, n = 3 from 3 independent experiments.
Figure 4.
qRT-PCR analysis of miRNA isolated with different kits.
A. Average CT values obtained for hsa-miR-191, hsa-miR-21 and ath-miR-159a on cDNA and pre-amplified cDNA (Pre-Amp) from 1 ml of plasma. Error bar = ±SEM, n = 9 from 3 independent experiments performed in triplicate. S/P = Serum/Plasma kit, CNA = Circulating Nucleic Acid kit. B. Average ΔCT values comparing the miRNeasy Serum/Plasma and CNA kit to the miRvana Isolation kit. ΔCT values calculated using the equation: Average miRNA CT using the miRvana kit - Average miRNA CT using another kit (i.e miRNeasy Serum/Plasma or CNA kit). Error bar = ±SEM, n = 9 from 3 independent experiments performed in triplicate. S/P = Serum/Plasma kit, CNA = Circulating Nucleic Acid kit. C. Two-tailed Spearman’s correlation analysis of ΔCT values obtained from TLDA A cards and samples extracted from 1 ml of plasma using the miRvana Isolation kit or 200 µl of plasma using the miRNeasy Serum/Plasma kit. Dotted lines represent the 95% confidence interval. Rho (ρ) and p-values are indicated on the graph. D. Average CT values obtained analysing hsa-miR-191, hsa-miR-21 and ath-miR-159a in cDNA produced from 1 ml, 500 µl or 200 µl of plasma using the miRNeasy Serum/Plasma kit. Error bar = ±SEM, n = 9 from 3 independent experiments performed in triplicate.
Figure 5.
Recovery of cfDNA and miRNA from plasma: effects of increasing centrifugation speed.
A. Quantitation of cfDNA using qPCR for GAPDH. Blood was processed within 2 hr or 6 hr post-venepuncture and cfDNA isolated from 1 ml of plasma. Whiskers of the box-plot show minimum and maximum values. B. Comparison of miRNA profiles from plasma processed 6 hr post-venepuncture compared to samples processed at 2 hr. Results presented as -ΔCT (change from 2 hours. Error bar = ±Std Dev, n = 5. *p<0.05; **p<0.01; ***p<0.001. C. Two-tailed Spearman’s correlation analysis of ΔCT values obtained from TLDA A cards for duplicate miRNA samples following centrifugation at 1000g and 2000g. Dotted lines represent the 95% confidence interval. D. Two-tailed Spearman’s correlation analysis of ΔCT values obtained from TLDA A cards for duplicate miRNA samples following centrifugation at 1000g and 10000g. Dotted lines represent the 95% confidence interval. E. Two-tailed Spearman’s correlation analysis of ΔCT values obtained from TLDA A cards for duplicate miRNA samples following centrifugation at 2000g and 10000g. Dotted lines represent the 95% confidence interval. For C - E, Rho (ρ) and p-values are indicated on each graph.