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

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.