围绕FFPE样本特性的DNA纯化纳米磁珠优化
Optimization of DNA purification nano beads specifically for FFPE samples
肖林 1吕晓芳 2曹师瑜 1江丹 3孙林雍 3李坤 2叶丰1
作者信息
- 1. 四川大学华西医院临床病理研究所,成都 610000
- 2. 四川大学化学学院,成都 610000
- 3. 四川大学华西医院病理科,成都 610000
- 折叠
摘要
目的 探讨围绕福尔马林固定石蜡包埋(formalin fixed paraffin embedded,FFPE)样本特性获取更高质量/得率的纳米磁珠核酸提取方案,改进分子病理技术.方法 合成4大类15个小类的备选磁珠,以FFPE样本为中心,筛选高质量/得率磁珠.模拟常规组织、粗针穿刺(肝脏)、纤维支气管镜样本(肺),装管相同张数连片.采用筛选的最佳磁珠与市场出售的常见磁珠试剂提取核酸,横向对比纯化总量、片段大小等质量参数.应用PCR和Sanger验证核酸的下游应用.结果 以FFPE样本DNA为中心筛选自制纳米磁珠,获得最佳性能纳米磁珠总回收率为58.5％±1.58％,5种市售商品化磁珠和3种国产磁珠总回收率为18.68％～40.71％.相同组织量(连片)提取的DNA总量,在模拟常规组织、粗针穿刺和纤维支气管镜样本核酸得率比市售试剂盒提高39.49％～181.72％(P＜0.05).模拟纤维支气管镜样本1张(4 μm)总量可达100 ng以上、5张总量可达400 ng以上.结论 以FFPE样本DNA为中心筛选的DNA纯化纳米磁珠,与商品化磁珠相比有较大提升,为临床分子病理检测的质量保证、自动化检测和项目拓展提供空间.
Abstract
Purpose Focusing on the characteristics of for-malin fixed paraffin embedded(FFPE)samples,explored nano-magnetic bead nucleic acid extraction solutions with higher qual-ity/yield and continued to improve molecular pathology technolo-gy.Methods Alternative magnetic beads were synthesised in four major categories and 15 sub-categories and we screened to obtain high-quality/yield magnetic beads centred on FFPE samples.Simulated conventional tissues,simulated coarse needle punctures(liver),and simulated fiberoptic bronchoscopy sam-ples(lungs)were sectioned with the same number of serial slices in tubes.The nucleic acids of slices were extracted using the best magnetic beads screened in this study and common com-mercially available kits,and then perform comparison and purifi-cation quality parameters such as total amount and fragment size.The downstream applications of nucleic acids were validated by PCR and Sanger sequencing.Results Screening all homemade nanomagnetic beads centered on the DNA of FFPE samples,the total recoveries of the best performance nanomagnetic beads were obtained to be 58.5％±1.58％,and the total recoveries of five commercially available commercial magnetic beads and three do-mestic kit magnetic beads ranged from 18.68％to 40.71％.The total amount of DNA(ng)extracted from the same amount of tis-sue(serial slices),the nucleic acid yield of this study in simu-lated conventional tissues,simulated coarse needle punctures,and simulated fiberoptic bronchoscopy samples were increased by 39.49％-181.72％compared with those of the commercially a-vailable kits(P＜0.05).The total amount of extracted nucleic acid from simulated fiberoptic bronchoscopy tissue sections can be more than 100 ng for 1 slice(4 µm)and more than 400 ng for 5 slices.Conclusion The DNA purification nanomagnetic beads screened with DNA from FFPE samples have a significant enhancement comparing to the existing commercial bead proto-cols,and provide space for quality assurance,automated testing,and program expansion for clinical molecular pathology testing.
关键词
石蜡包埋组织/DNA提取/纳米磁珠/PCR/SangerKey words
paraffin-embedded tissue/DNA extraction/nano-magnetic beads/PCR/Sanger引用本文复制引用
基金项目
四川大学华西医院成果转化项目(CGZH21011)
四川大学华西医院临床孵化项目(2020HXFH009)
出版年
2024
国家科技期刊平台
NSTL
万方数据