RESUMO
Cell-free DNA (cfDNA), a burgeoning class of molecular biomarkers, has been extensively studied across a variety of biomedical fields. As a key component of liquid biopsy, cfDNA testing is gaining prominence in disease detection and management due to the convenience of sample collection and the abundant wealth of genetic information it provides. However, the broader clinical application of cfDNA is currently impeded by a lack of standardization in the preanalytical procedures for cfDNA analysis. A number of fundamental challenges, including the selection of appropriate preanalytical procedures, prevention of short cfDNA fragment loss, and the validation of various cfDNA measurement methods, remain unaddressed. These existing hurdles lead to difficulties in comparing results and ensuring repeatability, thereby undermining the reliability of cfDNA analysis in clinical settings. This review discusses the crucial preanalytical factors that influence cfDNA analysis outcomes, including sample collection, transportation, temporary storage, processing, extraction, quality control, and long-term storage. The review provides clarification on achievable consensus and offers an analysis of the current issues with the goal of standardizing preanalytical procedures for cfDNA analysis.
RESUMO
Background: High-quality RNA extraction from tissue samples is of key importance for scientific research and translational medicine. Tissue collection and preparation may affect RNA quality. In this study, we investigated effects of warm ischemia time, cryopreservation, and grinding methods on RNA quality. Methods: Total RNA was extracted from mouse kidney tissues with warm ischemia times of 0, 30, 60, 90, and 120 minutes. Half of the tissues were used to extract RNA immediately, while the others were cryopreserved in the vapor phase of liquid nitrogen for 6 months before RNA extraction. A mortar, homogenizer, and tissue lyser were used to grind tissues. RNA was extracted by TRIzol, and RNA integrity was assessed by the RNA integrity number (RIN) value. Results: For fresh tissues and frozen tissues with warm ischemia time within 60 minutes, RIN values were above 7.0 and remained above 6.0 with warm ischemia time within 120 minutes. For the same warm ischemia time, RIN values of frozen tissues were slightly lower than those of fresh tissues. No significant RIN value alterations were observed among grinding methods, but for RNA extraction efficiency, a mortar was much less efficient than the homogenizer or tissue lyser. For frozen tissues, RNA tended to degrade within 8 minutes at room temperature. Conclusions: Mouse kidney tissues with a warm ischemia time within 120 minutes are suitable for general RNA-related research. For tissues with a warm ischemia time within 60 minutes, cryopreservation may not affect RNA quality. The duration of frozen tissues held at room temperature before grinding affects the integrity of RNA, while grinding methods do not affect RNA integrity.
