Patients and ethical declarations
We used 38 plasma samples from 38 patients, including patients with benign lung nodules and patients with stage I/II NSCLC. Clinical biospecimens were from the Johns Hopkins Lung Cancer Specialized Program of Research Excellence (SPORE) Baltimore, MD, and the University of Illinois at Chicago Hospital Health Science System (UIHHSS, Chicago, IL, USA).
The study was approved by the Institutional Review Board (IRB) of Johns Hopkins University (JHU) and the IRB of the University of Illinois at Chicago (UIC). The approval documents were NA_00005998 for JHU and IRB #2017-1286 and #2018-0755 for UIC. All procedures performed in our study involving human participants complied with the ethical standards of the National Research Committee and the Declaration of Helsinki of 1964 and subsequent amendments. All experimental protocols were approved before the start of the study. Informed consent was obtained from each patient, and peripheral blood was collected after obtaining informed consent and before patients underwent surgical resection or any treatment.
Whole peripheral blood (7.5 ml) was collected in an anticoagulant tube (K2EDTA) and poured very slowly into a 15 mL conical tube with 5 mL of Ficoll-Paque PLUS buffer (Millipore-Sigma, Cat #GE17-1440-02). The layered mixture was centrifuged for 10 minutes at 3000 rpm at 4°C, then the upper plasma layer was transferred to 1.5 ml tubes. Samples should not be processed if the red blood cells have been lysed.
Reagents and machines for RNA extracted from plasma
TRIzol Reagent (Thermo Fisher Scientific, Cat. No. 15596018), TRIzol LS Reagent (Ambion, Cat. No. 10296010), Chloroform (Sigma-Aldrich, Cat. No. C2432), Glycogen (Thermo Fisher Scientific, Cat. No. R0551), 3 M sodium acetate (pH5.2, Quality Biological, cat# 351035721), ethyl alcohol (Thermo Scientific Richard-Allan Scientific, for in vitro diagnostic use), isopropyl alcohol (Thermo Scientific Richard-Allan Scientific, for in vitro diagnostic vitro), nuclease-free water (Cell Signaling Technology, cat # 12931S), vortex mix (VWR, Analog Vortex Mixer), and centrifuge (Thermo Scientific, Legend Micro 21R). MagMAX Blood RNA Isolation Kit (ThermoFisher Scientific, Cat# AM1837), NucleoSpin for miRNA and RNA purification kit (Macherey-Nagel, Cat# 740971.50) and Plasma/Serum RNA Purification kit (Norgen Biotek Corp, Cat# 56100).
Urea polyacrylamide denaturing gel and silver stain
Ten microliters of sample was mixed with an equal volume of Gel Loading Buffer II (Thermo Fisher Scientific, cat # AM8546G), and small RNA marker (Abnova, cat # R0007) was used. The mixtures were heated at 95°C for 5 min to denature any secondary structure. Samples and markers were separated in 15% denaturing polyacrylamide gels (Invitrogen, cat # EC6885BOX) at 190 V for 50 min. Silver staining was performed using a SilverXpress silver staining kit (Thermo Fisher Scientific, Cat # LC6100) according to the manufacturer’s instructions.
Adapter ligation, RT and qPCR
The whole process for assessing pfeRNA expression levels was similar to that we previously described.4,7,17,25. Specifically, the method includes adapter ligation, RT, and QuantStudio PCR. An adapter with both 5′ and 3′ modifications ligates only the 3′ end of sncRNA and improves ligation efficiency. For each ligation reaction, 5 µl of total sncRNA and 1 µl (2 µM) of adapter were ligated using single-stranded truncated T4 RNA ligase 2 (New England Biolabs, cat # M0242 L) for one overnight at 16°C, and the ligation reaction was terminated at 65°C for 15 min. For RT, the SuperScript II first-strand synthesis system (Thermo Fisher Scientific, cat # 18064) and gene-specific reverse primers were used, and the total volume was 20 μl after RT. For QuantStudio PCR, a common reverse primer and primers specific to individual pfeRNAs were used. Each sample was tested in duplicate, and the total volume of each reaction was 20 µl. The amplification conditions were denaturation at 95°C for 15 s (15 min for the first cycle), annealing at 60°C for 20 s, extension at 72°C for 20 s and 40 cycles using a QuantStudio 3 machine (Applied Biosystem in Thermo Fisher Scientific). Each experiment was repeated three times independently. All primers and adapters are listed in Supplementary Table S2.