Brain Tumour Research Group, Institute of Clinical Neurosciences, University of Bristol, Bristol, UK.
Department of Cellular Pathology, North Bristol NHS Trust, Bristol, UK.
J Clin Pathol. 2018 Aug;71(8):695-701. doi: 10.1136/jclinpath-2017-204969. Epub 2018 Feb 20.
Histopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses.
Total RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes, and .
Reference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene.
This study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.
组织样本的组织病理学分析越来越多地被用作分子病理学转化研究中核酸的来源。本研究调查了神经胶质瘤和对照中枢神经系统（CNS）福尔马林固定石蜡包埋（FFPE）组织衍生 RNA 是否适合进行基因表达分析。
从对照（颞叶切除术组织）和神经胶质瘤 FFPE 组织样本中提取总 RNA。测定 RNA 纯度（260/280 比值）并进行 RNA 完整性数值（RIN）分析。随后将 RNA 用于 RT-qPCR 检测两个参考基因 和 的表达。
当使用从 FFPE 组织中提取的 RNA 时，对照和神经胶质瘤组织之间的参考基因表达是等效的，这对 CNS 基因表达研究的生物学归一化具有重要意义。对照和神经胶质瘤 FFPE 组织的平均 RIN 值存在显著差异。260/280 比值或 RIN 值与总 RNA 产量之间无显著相关性。组织块的年龄不影响 RNA 的产量、片段化或纯度。RIN 或 260/280 比值与任一参考基因的平均 qPCR 循环阈值之间均无显著相关性。
本研究表明，即使在储存 60 个月后，常规可用的 CNS FFPE 组织也适合 RNA 提取和下游基因表达研究。与神经胶质瘤和对照 FFPE 组织块相关的大量 RNA 片段化并未阻止下游 RT-qPCR 基因表达分析。需要对存档和前瞻性收集的 FFPE 标本进行交叉验证，以进一步证明 CNS 组织块可用于新的转化分子生物标志物研究。
