Molecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical Sciences, 27/33 Szpitalna St., 60-572, Poznan, Poland.
Respir Res. 2024 Nov 30;25(1):422. doi: 10.1186/s12931-024-03026-3.
Precision-cut lung slices (PCLS) are ex vivo models with preserved lung cell populations and maintained tissue architecture. PCLS are, therefore, a powerful tool in respiratory research to study molecular mechanisms that closely reflect whole tissue biology. High-quality RNA and protein extraction from PCLS is, however, challenging as agarose significantly interferes with the yield and purity of extracted material. The present study aimed to optimize QIAzol-based isolation protocol for high-yield and quality RNA, miRNA, and protein extraction from PCLS.
PCLS were prepared from 10 to 15-week-old Wistar rats and cultured for 7 days in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 0.1% FBS, penicillin, and streptomycin. LDH release to PCLS culture media was measured to determine cellular cytotoxicity. To select the optimal miRNA/RNA isolation protocol, we tested two different times (10 min, 2 h) and temperatures (room temperature, 4 °C, and -20 °C) of precipitation with isopropanol. Finally, we also assessed isolation with GHCL (guanidinium hydrochloride) extraction buffer. To select the optimal protein isolation protocol, we tested protein precipitation for 10 min at room temperatures (21 ± 1 °C) with 1.5 volumes of isopropanol and 3 volumes of acetone per 1 volume of phenol-ethanol supernatant. Additionally, we also tested protein precipitation for 3 h at -20 °C with 3, 5, and 7 acetone volumes per 1 volume of phenol-ethanol supernatant. We also validated protein precipitation with back extraction buffer instead of 100% ethanol. To measure the general efficiency of the optimized QIAZ-4 protocol, we used native rat lungs. PCLS for the ex vivo model of allergic inflammation were treated with IL-13 at a concentration of 80 ng/ml.
Standard QIAzol isolation protocol provided RNA, miRNA, and protein with low yield and poor quality. We found that 2-h isopropanol precipitation at 4 °C with a high concentration of salts significantly increased the yield and quality of extracted RNA and miRNA and provided acceptable qPCR efficiency (between 90 and 110%). Surprisingly, 2-h isopropanol precipitation at -20 °C significantly increased qPCR efficiency above the acceptable range (average efficiency: 120.4%). As for protein extraction, we found that 3-h acetone precipitation at -20 °C provided the highest yield with linear protein detection on Westen Blot. Optimized QIAZ-4 provided significantly higher miRNA and RNA yield compared to standard QIAzol protocols. We also found a significantly increased expression of Eotaxin-1 in PCLS treated with IL-13 as compared to the untreated controls.
In our study, we described a simple QIAzol-based method for the simultaneous isolation of RNA, miRNA, and protein from PCLS.
精密切割肺切片 (PCLS) 是一种具有保留的肺细胞群体和维持的组织结构的离体模型。因此，PCLS 是研究分子机制的呼吸研究的有力工具，这些分子机制非常接近整个组织生物学。然而，从 PCLS 中提取高质量的 RNA 和蛋白质具有挑战性，因为琼脂糖会严重干扰提取材料的产量和纯度。本研究旨在优化基于 QIAzol 的分离方案，以从 PCLS 中获得高产量和高质量的 RNA、miRNA 和蛋白质。
从 10 至 15 周龄的 Wistar 大鼠中制备 PCLS，并在补充有 0.1% FBS、青霉素和链霉素的 Dulbecco 改良 Eagle 培养基/营养混合物 F-12 (DMEM/F-12) 中培养 7 天。通过测量 LDH 释放到 PCLS 培养基中来确定细胞毒性。为了选择最佳的 miRNA/RNA 分离方案，我们测试了两种不同的异丙醇沉淀时间（10 分钟、2 小时）和温度（室温、4°C 和-20°C）。最后，我们还评估了使用 GHCL（盐酸胍）提取缓冲液的分离效果。为了选择最佳的蛋白质分离方案，我们测试了在室温（21°C ± 1°C）下用 1.5 体积异丙醇和 3 体积丙酮沉淀 10 分钟，相对于 1 体积酚-乙醇上清液。此外，我们还测试了在-20°C 下用 3、5 和 7 体积丙酮沉淀 3 小时。我们还使用回提缓冲液代替 100%乙醇验证了蛋白质沉淀。为了测量优化的 QIAZ-4 方案的一般效率，我们使用了天然大鼠肺。用 80ng/ml 的 IL-13 处理用于过敏性炎症的离体模型的 PCLS。
标准 QIAzol 分离方案提供的 RNA、miRNA 和蛋白质产量低、质量差。我们发现，4°C 下 2 小时异丙醇沉淀，高浓度盐显著提高了提取 RNA 和 miRNA 的产量和质量，并提供了可接受的 qPCR 效率（90%至 110%）。令人惊讶的是，-20°C 下 2 小时异丙醇沉淀显著提高了 qPCR 效率，超过了可接受范围（平均效率：120.4%）。至于蛋白质提取，我们发现，-20°C 下 3 小时丙酮沉淀提供了最高的产量，并且在 Western Blot 上线性检测到蛋白质。与标准 QIAzol 方案相比，优化的 QIAZ-4 提供了显著更高的 miRNA 和 RNA 产量。我们还发现，与未处理的对照相比，IL-13 处理的 PCLS 中 Eotaxin-1 的表达显著增加。
在本研究中，我们描述了一种简单的基于 QIAzol 的方法，可从 PCLS 中同时分离 RNA、miRNA 和蛋白质。
