Patients and ethics declarations
We used 38 plasma samples from 38 patients, including patients with benign pulmonary nodules and patients with stage I/II NSCLC. The 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 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 were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments. All experimental protocols were approved prior to study initiation. Informed consent was obtained from each patient, and peripheral blood was collected after informed consent was obtained and prior to the patients undergoing surgical resection or with any treatment.
Plasma preparation
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 Ficoll-Paque PLUS buffer (Millipore-Sigma, Cat #GE17-1440-02). The layered mixture was centrifuged for 10 min at 3000 rpm at 4 °C, and then the top plasma layer was transferred to 1.5 ml tubes. The samples should not be processed if the red cells had been lysed.
Reagents and machines for RNAs extracted from plasma
TRIzol reagent (Thermo Fisher Scientific, cat# 15596018), TRIzol LS Reagent (Ambion, Cat# 10296010), chloroform (Sigma-Aldrich, Cat No. C2432), glycogen (Thermo Fisher Scientific, cat# 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 use), nuclease-free water (Cell Signaling Technology, cat# 12931S), mixture vortex (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).
Denaturing urea polyacrylamide gel and silver staining
Ten microliters of sample were mixed with an equal volume of gel loading buffer II (Thermo Fisher Scientific, cat# AM8546G), and the small RNA marker (Abnova, cat# R0007) was used. The mixtures were heated to 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.
Adaptor ligation, RT, and qPCR
The whole process of evaluating pfeRNA expression levels was similar to that we described before4,7,17,25. Specifically, the method includes adaptor ligation, RT, and QuantStudio PCR. An adaptor with both 5′ and 3′ modifications ligates only the 3′-end of sncRNA and enhances the ligation efficiency. For each ligation reaction, 5 µl total sncRNAs and 1 µl (2 µM) adaptor were ligated using single-strand truncated T4 RNA ligase 2 (New England Biolabs, cat# M0242 L) 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 for individual pfeRNAs were used. Each sample was tested in duplicate, and the total volume of each reaction was 20 µl. 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 independent times. All primers and adaptors are listed in Supplementary Table S2.
