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 c....

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.

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