Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues

RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies.石蜡包埋组织（Paraffin-Embedded Tissues, PET）的RNA与DNA分析，是疾病表征、生物标志物探索及治疗方案筛选的重要诊断手段。由于福尔马林固定石蜡包埋（Formalin-Fixed and Paraffin-Embedded, FFPE）组织来源的核酸因生物分子发生化学修饰，在分子分析中的应用受限，因此无福尔马林的固定试剂（如PAXgene Tissue系统）的关注度日益提升。此外，生物医学研究与生物标志物开发高度依赖医学档案或生物库中长期储存的PET样本，以关联分子特征与长期疾病转归。 为此本研究开展对照实验，评估不同温度下长期储存FFPE与PAXgene组织固定石蜡包埋（PAXgene Tissue-fixed and Paraffin-Embedded, PFPE）组织对核酸稳定性及PCR实用性的影响。本研究选取常规临床场景下匹配的FFPE与PFPE人体组织，以及高度可控动物模型来源的大鼠组织，分别置于室温、4℃储存；其中动物组织额外设置-20℃与-80℃冷冻储存组。研究团队每隔固定时段提取RNA与DNA，最长随访时长达9年，并检测核酸完整性，以及其在定量逆转录PCR（Quantitative Reverse Transcription Polymerase Chain Reaction, RT-qPCR）或定量PCR（Quantitative Polymerase Chain Reaction, qPCR）检测中的实用性。 结果显示，室温储存的PET样本可出现核酸降解，4℃储存可延缓该降解过程，而-20℃或-80℃冷冻储存则可完全抑制降解。核酸降解与扩增子长度依赖性的RT-qPCR及qPCR效率降低显著相关。4℃储存可显著提升RT-qPCR与qPCR的扩增效能。无论储存时长与温度如何，人体与大鼠组织来源的PFPE样本所提取的化学未修饰核酸，其表现均优于FFPE样本。 综上，采用非交联固定剂并在低温（如4℃）下储存PET样本，可显著提升其分子分析效能，该方案应在前瞻性临床研究中予以考量。