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All procedures involving human tissue samples and clinical specimens in this study were performed in compliance with the institutional guidelines and approved by the Ethics Committee of Jilin Cancer Hospital (Ethical approval No. 202507-002-01). Written informed consent was obtained from all participants or their legal guardians prior to sample collection. Peripheral blood samples (10 mL per patient) were collected from 19 patients with advanced non-small cell lung cancer (NSCLC) at Jilin Cancer Hospital, yielding a total of 133 RNA samples. To ensure laboratory safety, all operations involving hazardous chemicals were performed in a biological safety cabinet.
Detailed information on the instruments, reagents, and consumables used in the experiment was provided in the Table of Materials.
Isolation of PBMC
Peripheral blood samples were processed for PBMC isolation within 2 h after collection. PBMCs were isolated and purified by Ficoll-Paque density gradient centrifugation at 400 × g for 25 min at room temperature with the brake disabled. After centrifugation, the intermediate milky white lymphocyte layer (containing PBMCs) was carefully aspirated16. Importantly, we strictly standardized the PBMC input concentration for each sample and each group to ensure consistent cell numbers across all experimental conditions, thus minimizing variation caused by cell input and processing. This ensures that the observed differences reflect genuine effects of different preservation methods rather than technical variability. For each patient, isolated PBMCs were divided into 7 tubes: one tube was used for immediate RNA extraction, and the concentration, purity, and integrity were assessed. The remaining six tubes were cryopreserved at a cooling rate of -1 ˚C/min: three were treated with 1 mL of 90% FBS + 10% DMSO, and the other three with 1 mL of TRIzol, followed by storage at -80 °C. RNA extraction and quality assessment were performed after 1, 3, and 6 months of frozen storage.
RNA extraction
Samples were removed from -80 °C storage and thawed at 4 °C until fully thawed17. Samples preserved with 90% FBS + 10% DMSO or TRIzol cryopreservation solution were washed once with PBS, centrifuged at 200 × g for 5 min at 4 °C, and the supernatant was discarded to retain the cell pellet. Total RNA was extracted using TRIzol. Specifically, TRIzol reagent was added to the microcentrifuge tube containing the processed sample, mixed by inversion, and incubated at room temperature for 5 min to ensure complete lysis. Subsequently, 200 µL of chloroform was added, mixed thoroughly by shaking, and incubated at room temperature for 15 min. After centrifugation at 2400 × g for 15 min at 4 °C, the upper aqueous phase was carefully aspirated and transferred to a new Eppendorf tube. Isopropanol (0.5 mL) was then added, mixed well, and incubated at room temperature for 5–10 min. Following centrifugation at 2400 × g for 10 min at 4 °C, the supernatant was discarded, leaving the RNA precipitate at the bottom of the tube.
The precipitate was resuspended in 75% ethanol (1 mL) with gentle shaking, followed by centrifugation at 1600 × g for 5 min at 4 °C. The supernatant was discarded, and the RNA pellet was allowed to air-dry at room temperature for 5–10 min. Finally, the RNA samples were dissolved in 50 µL DEPC-treated water and incubated in a 55–60 °C water bath for 5–10 min18. Detection of RNA concentration and purity. RNA concentration and purity were measured using the ultraviolet absorbance method with a multi-sample microvolume UV-Vis spectrophotometer (measurement range: 2.5–3000 ng/µL). RNA concentration was determined based on the absorbance value at 260 nm, while RNA purity was assessed using the A260/A280 ratio. The measurement software was launched; “Nucleic Acid” was clicked on the main interface. The detection platform was rinsed twice with double-distilled water, then 1.5 µL of the RNA sample was pipetted onto it. RNA-40 was selected for the sample type, “Measure” was clicked, and the concentration and A260/280 purity values were recorded. An A260/A280 ratio between 1.9 and 2.1 indicated high RNA purity, whereas values below 1.8 suggested protein contamination, and values greater than 2.2 indicated potential RNA degradation or residual isothiocyanate19.
RNA integrity detection
RNA integrity (RIN) was evaluated using an automated microfluidic electrophoretic bioanalyzer and detection kit. The RIN ranges from 1 to 10, with 1 indicating highly degraded RNA and 10 indicating intact RNA. For analysis, 1.2 µL of RNA was placed in a PCR tube, thermally denatured at 70 °C for 2 min using a thermal cycler, and immediately placed on ice. Subsequently, 1 µL of denatured RNA was added to the detection wells of the chip for analysis20. The software was launched, "Assays" was selected, and "RNA" was chosen from the dropdown menu. The program "Eukaryote Total RNA Nano Series II.xsy" was selected according to the reagent used. "Start" was clicked to initiate detection. After approximately 10 min, the peak number and peak height of the ladder were checked for normality.
Statistical methods
Normality tests were performed for all data. Normally distributed data were expressed as mean ± standard deviation (SD), while non-normally distributed data were expressed as median (interquartile range, IQR). Comparisons between two groups were analyzed using the paired t-test for normally distributed data and the related-samples nonparametric test for non-normally distributed data. For comparisons among three or more groups, repeated-measures analysis of variance was used for normally distributed data, and the related-samples nonparametric test was used for non-normally distributed data. All statistical analyses were performed using SPSS 19.0 and GraphPad Prism 9.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.