Effects on RNA Quality of PBMCs Under Different Cryopreservation Times and Processing Methods in Patients with Advanced Non-Small Cell Lung Cancer

Summary

This study compares the Triple Isolation Reagent (TRIzol) and the classical solution (FBS + DMSO) for cryopreserving NSCLC patients' PBMCs. TRIzol maintains satisfactory RNA integrity for up to 6 months, making it recommended for high-quality RNA preservation.

Abstract

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in RNA sequencing (RNA-seq), and the quality of their RNA directly influences test outcomes. However, cryopreservation can readily lead to RNA degradation in PBMCs, and the issue of how to improve RNA quality during long-term cryopreservation remains unresolved. PBMCS from fresh blood samples collected from patients with advanced non-small cell lung cancer (NSCLC) were utilized as controls. The RNA preservation efficacy of PBMC samples was compared between the classical cell cryopreservation solution (90% fetal bovine serum [FBS] + 10% dimethyl sulfoxide [DMSO]) and the common RNA extraction reagent (TRIzol) at -80 ˚C for 1, 3, and 6 months. RNA concentration, purity, and integrity (as measured by the RNA integrity number [RIN]) were assessed using a multi-sample microvolume UV-Vis spectrophotometer and an automated microfluidic electrophoretic bioanalyzer to provide a theoretical foundation for the preservation of high-quality RNA samples. This study demonstrated that the concentration, purity, and integrity of RNA in PBMCs decreased with extended cryopreservation duration. While the two processing methods had no effect on RNA concentration, they did affect RNA purity and integrity. The RNA integrity of PBMCs cryopreserved with TRIzol for 6 months remained satisfactory. Our results indicate that TRIzol contributes to effective cryopreservation and maintains high-quality RNA.

Introduction

RNA-seq of PBMC in NSCLC patients plays a critical role in evaluating the efficacy of immunotherapy. Non-small cell lung cancer (NSCLC) is a common malignant tumor worldwide, and about 70% of patients are diagnosed at an advanced stage with a poor prognosis^1,2. Immunotherapy has revolutionized the treatment paradigm for NSCLC, providing innovative therapeutic strategies^3,4. In the treatment of immune checkpoint inhibitors (ICI), RNA sequencing (RNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from NSCLC patients can monitor the dynamic changes of clinical benefits of immunotherapy⁵. Sample sources for RNA-seq include tumor tissue and PBMCs^6,7, with PBMCs serving as a vital biological specimen in both clinical trials and basic scientific research⁸. Cryopreservation represents the primary method for long-term sample storage, maintaining cells and tissues in a quiescent state while preserving their biological potential and characteristics. Consequently, cryopreservation of PBMCs has emerged as a feasible and widely adopted approach. Cryopreserved PBMC facilitates batch analysis of samples, minimizes intra-laboratory and inter-laboratory variability, and optimizes resource utilization^8,9. For instance, studies have shown that using a frozen RPMI-1640 medium supplemented with 40% fetal calf serum (FCS) and 20% dimethyl sulfoxide (DMSO) for PBMC cryopreservation demonstrates that the proportions of T and B cells expressing the combination of CD39 and CD73 remained relatively stable within six months of storage¹⁰. One study compared three commercial cryopreservation kits and two cryoprotective agents (90% RPMI-10% DMSO, 90% plasma-10% DMSO), revealing similar cell viability (81.0%) and recovery rates (73.7%) across methods. However, all cryopreserved PBMCs exhibited significantly reduced viability compared to fresh samples⁵. Further research demonstrated that resuspended PBMC precipitates in TRIzol reagent and immediately froze them at -80 °C. After 1–2 days, they observed that the RIN value of PBMCs was higher than that of whole blood samples¹¹.

Another investigation compared leukocyte samples treated with TRIzol, RNAlater, and other reagents, showing that all methods achieved RNA integrity number (RIN) values >7. RNA integrity was assessed using an automated microfluidic electrophoretic bioanalyzer, and RIN was calculated as a quantitative measure of RNA quality. Samples with RIN ≥7 were considered suitable for RNA‑seq analysis¹². Notably, the leukocyte + RNAlater group exhibited significantly lower RNA yield compared to the leukocyte + TRIzol group¹². However, current research on PBMC cryopreservation predominantly focuses on cell viability and immune cell phenotype analysis post-thaw; limited attention has been devoted to systematic, long-term assessment of RNA quality under different preservation conditions. Moreover, standardized protocols for cryopreserving PBMC samples to preserve RNA integrity remain underdeveloped^13,14,15. This gap is particularly relevant for NSCLC immunotherapy, where high-quality RNA from longitudinally collected PBMCs is essential for reliable biomarker discovery and monitoring of treatment response.

In this study, we hypothesize that the choice of cryopreservation method significantly influences the long-term integrity and quality of RNA derived from PBMCs, and an optimized protocol can maintain RNA suitability for sensitive assays such as RNA-seq. To address this, freshly isolated PBMCs from blood samples collected from advanced NSCLC patients were used as controls. The RNA preservation efficacy of PBMC samples was compared between two treatment methods (90% FBS + 10% DMSO and TRIzol) at -80 °C for 1, 3, and 6 months (Figure 1). RNA concentration, purity, and integrity (as measured by the RIN) were evaluated using a multi-sample microvolume UV-Vis spectrophotometer and an automated microfluidic electrophoretic bioanalyzer. This study aims to provide a theoretical foundation for high-quality RNA sample preservation and to advance translational research and sample preservation in tumor precision medicine.

Protocol

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.

Results

Effects of different cryopreservation times and processing methods on RNA concentration in PBMC samples
The effect of different cryopreservation times: The fresh PBMC samples from advanced NSCLC patients served as the control group. The samples were treated with either TRIzol or 90% FBS + 10% DMSO and stored at -80 °C for 1, 3, and 6 months; total RNA was extracted to measure RNA concentration. For TRIzol-treated samples, RNA concentrations across the four groups were as follows: fresh 523.5 (485.0) ...

Discussion

Many factors influence RNA quality in biological samples, including pre-analytical variables, transport conditions, duration of processing at ambient temperature, tissue necrosis, temperature and freezing products, aliquot size and number, as well as storage duration have a significant impact on RNA quality²¹. For identical samples, variations in cryopreservation time and processing methods can significantly affect RNA quality. Previous studies have highlighted the critical importance of RNA integ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agilent 2100 Bioanalyzer	Agilent	G2939B	Detection of RNA integrity, evaluating RNA quality by generating the RNA Integrity Number (RIN)
Agilent RNA 6000 Nanokit	Agilent	5067-1511	In conjunction with the Agilent 2100 Bioanalyzer, providing dedicated reagents and chips for RNA integrity
Centrifuge	Eppendorf	5424R	For extracting RNA
Centrifuge	Eppendorf	5810R	For extracting PBMC
Chloroform	China National Pharmaceutical Group Corporation	20191031	For RNA extraction by the Trizol method, used to separate the aqueous phase containing RNA from the organic phase containing impurities
Dimethyl sulfoxide	Beijing Dingguo Changsheng	DH105-2	As a cryopreservation solution for PBMCs, enabling long-term storage of samples at -80 °C
Tube	Corning	MCT-150-C	For sample storage, reagent mixing, centrifugation, and other experimental operations
Ethanol	Shandong Lierkang	25071901A	IIn RNA extraction by the Trizol method, used for washing RNA precipitates to remove residual
Fetal bovine serum	Xpbiomed	C04001-500	As a cryopreservation solution for PBMCs, enabling long-term storage of
Ficoll separation solution	Beijing Dingguo Changsheng Biotechnology CO., LTD	CC0161	For isolation and purification of PBMC via density gradient centrifugation
GeneTouch(Plus) Thermal cycler	Bioer	TC-EA-48DA	For thermal denaturation of RNA (70 °C for 2 min), followed by detection with the bioanalyzer
Isopropanol	China National Pharmaceutical Group Corporation	20210929	In RNA extraction by the Trizol method, used for RNA precipitation
Nanodrop 8000 Spectrophotometer	GeneCompany	ND-8000	For RNA concentration (based on absorbance at 260 nm) and purity (based on A260/A280 ratio) using the ultraviolet absorbance method
PCR Tubes	Corning	PCR-02-C	For thermal denaturation of RNA (70 °C for 2 min), followed by detection with the bioanalyzer
Phosphate-buffered saline	Procell system	PB180327	For direct lysis and preservation of PBMCs, or subsequent total RNA
Triple Isolation Reagent	Adsin	ads60154	For direct lysis and preservation of PBMCs, or subsequent total RNA