Legend to Supplemental Tables
Supplemental Table S1. Details on the tests performed to optimize circulating miRNA
detection by RT-qPCR.
Sheet 1 – S1A. The table shows data on the 48 miRNA assays used in this study and the
results of tests described in Fig.2A. Shown are the names of the miRNA assay, the type of
miRNA selected [endogenous house-keeping reference miRNA (HK miRNA); lung cancer
diagnostic miRNA (Lung miRNA), Spike-in miRNA (Spike), negative control (NEG CTRL),
other known circulating miRNAs related to non-lung diseases plus two small RNAs typically
poorly detected in the serum (other)], the results of the Reverse Transcription Test (TEST 1;
Cq of detection with (RT-PreAMP) or without (RT) the pre-amplification step, N. of
replicates = 4), and the results of the 8-point dilution linearity test (TEST 2).
Based on the test results, 4 miRNAs did not fulfill the detection requirement (Cq>30 in Test
#1) and 6 miRNAs did not show optimal sensitivity in 8-point linearity test (R2 <0.90 in Test
#2). These miRNAs were marked as “failed” and were excluded from further analyses.
Sheet 2 – S1B. The table shows the results of the analysis performed on the precision of
detection of miRNAs of the miR-Test [6 HK miRNAs (HK miRNA), 13 lung cancer
diagnostic miRNAs from the miR-Test (13-miRNA)] described in Figures 2C-H. For each
miRNA, we report the average variance calculated over replicates of qPCR, RT-AMP or
Extraction. Extraction replicas were performed on the same day (intra-assay precision,
INTRA), or in four different days (inter-assay precision, INTER). N, number of replicas for
each test. S, number of different sera used.
Supplemental Table S2. Details on the experiments investigating hemolysis
Sheet 1 – hemolysis curve. The table shows data relating to the samples used to generate the
‘hemolysis curve’ along with the ratios between miR-146a and miR-15b, miR-19a and miR19b, used to calculate the “miRNA hemo sensor”.
Sheet 2 – RT-qPCR data of the hemolysis curve
The table shows the raw data related to the miR-Test miRNAs in the hemolytic curve (Fig.4).
Shown also is the correlation between miRNA levels (Cq) and the % of hemolysis by
Pearson. miRNAs with a Pearson correlation coefficient > 0.8 are marked.
Supplemental Table S3. Details on the measurement of circulating miRNAs in the
clinical cohort (COSMOS)
The table shows data on the measurement of circulating miRNAs in the clinical COSMOS
cohort (N = 972) (14). Reported are the details of the extraction (date and the round) and RT
and Pre-AMP reactions for the samples along with the values of: the negative control (celmiR-39), the spiked-in miRNAs, the mean of the 6 endogenous HK reference miRNAs (6HK), the mean of the 13 miRNAs of the diagnostic model. The details of the hemolysis
analysis score (hemo index, hemo group and miRNA hemo sensor) and the risk score based
on the miR-Test are also reported.
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