Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.
J Vet Diagn Invest. 2024 Jan;36(1):70-77. doi: 10.1177/10406387231211908. Epub 2023 Nov 28.
We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue ( = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and -tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.
我们评估了福尔马林固定时间和包含周围组织对福尔马林固定、石蜡包埋（FFPE）尿路上皮癌（UC）组织中 microRNA（miRNA）循环定量（Cq）值的影响（ = 3），并评估了这些条件对 1 只健康犬尿液中 miRNA 的影响。使用商业试剂盒提取 miRNA，并使用 miRNA 特异性荧光计在正常膀胱组织卷、UC 组织芯和来自 4 个 FFPE 膀胱切片的膀胱肌肉组织芯（3 个 UC，1 个正常）中定量，外加 1 个 UC 在福尔马林固定 1、8、15 和 22 d 后再进行石蜡包埋。从 1 只健康犬收集尿液 4 次；每次取 1 mL 等分试样，在 20、4、-20 和-80°C 下分别储存 4、8、24 和 48 h，以及 1 和 2 周。对于 FFPE 组织和尿液，我们使用逆转录定量实时 PCR（RT-qPCR），在每个组织或样本中定量 miR-143、miR-152、miR-181a、miR-214、miR-1842 和 RNU6B，使用 miR-39 作为外源性对照基因。使用方差分析和 -检验比较 Cq 值。福尔马林固定时间不会改变 miRNA Cq 值；包含肌肉层会导致膀胱组织中 miR-152、miR-181a 和 RNU6B 的 miRNA Cq 图谱在统计学上存在差异。无细胞尿液中的 miRNA 无论储存温度如何，在长达 2 周内都是稳定的。我们的研究结果支持使用储存的 FFPE 和尿液样本进行 miRNA 检测；我们建议仅在 FFPE 切片中的感兴趣组织中测量 miRNA。
