Supplementary Fig. 1. No significant difference was observed in the plasma samples
prepared on day 1 for Ct values of miR-16 (P = 0.464) and RNU6B (P = 0.418) between
20 cancer patients and 20 healthy controls.
Supplementary Fig. 2. miR-16 displays higher stability and abundance than does
RNU6B in plasma. A. Ct values of miR-16 did not change after prolonged storage at 4°C
for 7 days (P > 0.05). B. RNU6B exhibited less stable than miR-16 illustrated by that the
Ct values of RNU6B in the samples stored at 4°C for 7 days were significantly higher
than that in the samples prepared on day 1 (P = 0.03). Furthermore, mean Ct value of
miR-16 was statistically lower than that of RNU6B in the same samples tested on day 1
(19.2 ± 1.2 vs. 29.6 ± 2.9, P<0.05).
Supplementary Fig. 3. Stability of endogenous miRNAs in archived plasma samples.
Aliquots of plasma specimens were stored at -20°C for 1, 3, 7, and 30 days. A.
Bioanalyzer showed that there was increasing degradation of total RNA demonstrated by
gradually decreased heights of 18S and 28S peaks from day 1 to day 30. B. There was no
effect on expression levels of the miRNAs determined by qRT-PCR in the same
specimens. The figure only shows the results for miR-21, 126, 182, 210, 486-5p, and 375.
Supplementary Fig. 4. The endogenous plasma miRNAs have substantial resistance to
the enzymatic cleavage of RNase A. Expressions of the miRNAs were measured in the
plasma samples treated with RNase A at different concentrations and the samples without
the treatment. The abundance of the miRNAs in plasma with the different treatments did
not display significant difference (All P>0.05) (A-F). The figure only shows the results
for miR-21, 126, 182, 210, 486-5p, and 375. However, synthesized cel-miR-238 spiked
in the same specimens treated with or without RNase A was almost not detected (G).
Supplementary Fig. 5. Sensitivity and dynamic range of qRT-PCT assay for quantification of
synthesized cel-miR-238 in plasma. Error bars represent standard deviation to reflect the
variations among the replicate measurements.
Supplementary Fig. 6. QRT-PCR assay is highly specific and could discriminate miRNAs that
are different members of the same human miRNA gene family, miR-486. The synthetic miR486-5p or miR-486-3p was subjected to two independent qRT-PCR reactions, where in each
reaction there were PCR primers specific to only one of the two miR-486 variants. Non-specific
amplification was found, however amplification only of the appropriate gene matching the
specific primers was observed. Error bars represent standard deviation to reflect the variations
among the replicate measurements.
Supplementary Fig. 7. miRNAs are accurately and robustly measured by qRT-PCR.
RNA isolated from plasma was diluted in DEPC water by ten orders of magnitude,
respectively. The serially diluted RNAs served as experimental samples for measuring
expression of each miRNA by qRT-PCR. There was excellent linearity between the RNA
input and the Ct values for the qRT-PCR assay. The assay had a dynamic range of at least
eight orders of magnitude, and was capable of detecting as few as 10 copies of miRNA
per PCR reaction, and the correlation coefficient was at least 0.992 for the miRNAs
tested. The figure only shows the results for miR-21, 145, 205, 375, 486-5p, and 429.
Error bars represent standard deviation to reflect the variations among the replicate
measurements.
Supplementary table 1. Expression levels of the five miRNAs in plasma of
an independent of cohort of 58 NSCLC patients and 29 healthy individuals
MiRNAs
58 cancer cases
28 healthy smokers
P
Mean ± SD
Mean ± SD
miR-21
6.647 ± 2.045
0.628 ± 0.092
0.0002 *
miR-182
6.783 ± 2.259
2.195 ± 0.673
0.0006 *
miR-210
2.651 ± 0.772
1.126± 0.558
0.0005 *
miR-126
4.598 ± 1.323
8.189 ± 2.149
0.0003 *
miR-486-5p
3.659 ± 0.964
8.852 ± 2.609
0.0001 *
Abbreviations: NSCLC, non-small-cell lung cancer; SD, standard deviation.
* Statistical significance of expression levels of the miRNAs between
NSCLC patients and healthy subjects. P < 0.05.
Supplementary table 2. Capability of the five miRNAs to discriminate
NSCLC patients from healthy subjects *
miRNAs
AUC (SE)
Sensitivity %
Specificity %
miR-21
0.816 (0.07)
79.31
65.52
miR-486-5p
0.878 (0.05)
84.48
68.97
miR-126
0.760 (0.06)
68.97
82.76
miR-210
0.746 (0.04)
74.14
68.97
miR-182
0.662 (0.05)
51.72
75.86
* Receiver-operator characteristic (ROC) curve and the area under ROC
curve (AUC) analyses were applied to determine optimal thresholds that
define expression levels of the tested genes, yielding corresponding
maximum sensitivity and specificity of each gene in distinguishing NSCLC
from healthy individuals.
Abbreviations: NSCLC, non-small-cell lung cancer; SE, standard error.