Research Article

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

DOI:

10.3791/70261

April 21st, 2026

* These authors contributed equally

In This Article

Summary

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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

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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

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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 prognosis1,2. Immunotherapy has revolutionized the treatment paradigm for NSCLC, providing innovative therapeutic strategies3,4. 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 c....

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Protocol

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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.

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Results

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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) .......

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Discussion

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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.......

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Disclosures

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The authors declare no conflict of interest.

Acknowledgements

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We gratefully acknowledge and thank participants for their help with the experiment and for their support of the fund. This study was supported by the grants from Jilin Provincial Department of Science and Technology (Grant No.YDZJ202501ZYTS691), Health Commission of Jilin Province (Grant No.2023jc064).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Agilent 2100 BioanalyzerAgilentG2939BDetection of RNA integrity, evaluating RNA quality by generating the RNA Integrity Number (RIN)
Agilent RNA 6000 NanokitAgilent5067-1511     In conjunction with the Agilent 2100 Bioanalyzer, providing dedicated reagents and chips for RNA integrity
Centrifuge                             Eppendorf  5424RFor extracting RNA
Centrifuge                      Eppendorf  5810RFor extracting PBMC
Chloroform                 China National Pharmaceutical Group Corporation20191031For RNA extraction by the Trizol method, used to separate the aqueous phase containing RNA from the organic phase containing impurities
Dimethyl sulfoxideBeijing Dingguo ChangshengDH105-2As a cryopreservation solution for PBMCs, enabling long-term storage of samples at -80 °C
TubeCorningMCT-150-CFor sample storage, reagent mixing, centrifugation, and other experimental operations
EthanolShandong Lierkang25071901AIIn RNA extraction by the Trizol method, used for washing RNA precipitates to remove residual
Fetal bovine serumXpbiomedC04001-500As a cryopreservation solution for PBMCs, enabling long-term storage of
Ficoll separation solutionBeijing Dingguo Changsheng Biotechnology CO., LTDCC0161For isolation and purification of PBMC via density gradient centrifugation
GeneTouch(Plus) Thermal cyclerBioerTC-EA-48DAFor thermal denaturation of RNA (70 °C for 2 min), followed by detection with the bioanalyzer
IsopropanolChina National Pharmaceutical Group Corporation20210929In RNA extraction by the Trizol method, used for RNA precipitation
Nanodrop 8000 SpectrophotometerGeneCompanyND-8000For RNA concentration (based on absorbance at 260 nm) and purity (based on A260/A280 ratio) using the ultraviolet absorbance method
PCR TubesCorningPCR-02-CFor thermal denaturation of RNA (70 °C for 2 min), followed by detection with the bioanalyzer
Phosphate-buffered saline Procell systemPB180327For direct lysis and preservation of PBMCs, or subsequent total RNA
Triple Isolation Reagent Adsinads60154For direct lysis and preservation of PBMCs, or subsequent total RNA

References

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  1. Ettinger, D. S., et al. NCCN guidelines insights: Non-small cell lung cancer, version 1.2020. J Natl Compr Canc Netw. 17 (12), 1464-1472 (2019).
  2. Siegel, R. L., Miller, K. D., Jemal, A. Cancer statistics, 2020. CA Cancer J Clin. 70 (1), 7-30 (2020).
  3. Reck, M.,....

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Tags

PBMC CryopreservationRNA IntegrityRNA ExtractionTRIzol PreservationFetal Bovine SerumDimethyl SulfoxideMicrofluidic ElectrophoresisUV Vis Spectrophotometry

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