新型超声快速处理活检标本保存不同年限对DNA质量的影响  

Effects of different storage durations on DNA quality of biopsy specimens with novel ultrasound processing

作　　者：石晨曦[1] 朱卫东[1] 李三恩 李秀明 师逢 丁亚云[1] Shi Chenxi;Zhu Weidong;Li Sanen;Li Xiuming;Shi Feng;Ding Yayun(Department of Pathology,First Affiliated Hospital of Soochow University,Suzhou 215000,Jiangsu Province,China;Department of Dermatology,Suzhou Municipal Hospital,Suzhou 215000,Jiangsu Province,China)

机构地区：[1]苏州大学附属第一医院病理科,江苏省苏州市215000 [2]苏州市立医院本部皮肤科,江苏省苏州市215000

出　　处：《中国组织工程研究》2025年第13期2655-2660,共6页Chinese Journal of Tissue Engineering Research

基　　金：苏州市科技局项目(2021YFA1100600),项目负责人:师逢;苏州大学附属第一医院自然科学基金博习培育计划(2018YFA0107500),项目负责人:丁亚云;南京医科大学科技发展基金(NMUB20220189),项目负责人:师逢。

摘　　要：背景:新型超声组织处理技术越来越多地被用来进行分子生物学分析,研究新型超声处理不同存储年限组织DNA的质量,对进一步分子检测的标本质控具有重要意义。目的:探讨新型超声处理活检标本存储不同年限对DNA质量的影响,以期为分子检测探索最佳的标本存储时间。方法:收集40例乳腺穿刺小活检组织,采用超声技术制作石蜡标本,按照存储年限分为4组:<1年组、1-3年组、>3-5年组及>5年组,每组10例,对石蜡标本进行切片,每张切片厚3μm,切片10-15张,提取DNA后通过Nanophotometer N60超微量分光光度计和Qubit 4.0荧光计检测DNA的质量浓度,记录A_(260)/A_(280)比值判定DNA的纯度,利用全自动毛细管电泳核酸分析仪(Qsep 100)检测DNA片段完整性,以评估DNA片段的质量。结果与结论:4组样本A_(260)/A_(280)均值在1.8-2.0之间,达到纯度要求,无明显差异。4组样本的DNA质量浓度(Qubit浓度)均值分别为30.39,14.33,2.52,1.95 ng/μL;DNA的平均N/Q比值分别为6.48,14.18,24.56,29.86;DNA质量数均值分别为5.64,1.76,1.24,0.80;大片段占比均值分别为56.08%,17.72%,12.68%,7.90%。PCR检测内控基因Ct均值分别为15.32,17.09,18.39,21.24。与<1年组相比,其余3组DNA浓度显著降低,N/Q比值显著增加,DNA质量数和大片段占比均值显著降低,Ct值升高,差异有显著性意义(P<0.05)。实验结果表明,对于新型超声处理活检标本,应优先选择存储<1年的样本进行日常分子检测,储存3年内的样本可满足二代测序等检测要求,5年内样本仅可尝试进行PCR等检测,存储超过5年的样本不建议进行后续分子检测。BACKGROUND:The technique of ultrasound processing is widely used for molecular biological analysis.It is of great significance to study the DNA quality of tissue with different storage years under new ultrasonic treatment for further specimen quality control of molecular detection.OBJECTIVE:To explore the effects of different storage durations on DNA quality in specimens with ultrasound processing to investigate the optimal storage time for molecular tests.METHODS:Forty specimens of breast biopsy were collected and paraffin specimens were prepared by ultrasonography.These specimens were divided into four groups based on their storage periods:<1 year,1-3 years,>3-5 years,and>5 years,which contained 10 cases in each group.Paraffin specimens were sliced;each slice was 3μm thick;10-15 slices were taken,and DNA was extracted.The mass concentration of DNA was examined by Nanophotometer N60 ultra-micro spectrophotometer and Qubit 4.0 fluorometer.The purity of the DNA was analyzed by the ratio of A_(260)/A_(280).DNA fragment integrity was measured by capillary electrophoresis(Qsep 100)to evaluate the quality of the DNA fragments.RESULTS AND CONCLUSION:The mean values of A_(260)/A_(280) in the four groups were between 1.8 and 2.0,meeting the requirements of tests,without significant differences.The mean values of DNA mass concentration(Qubit concentration)were 30.39,14.33,2.52,and 1.95 ng/μL,respectively.The mean values of the N/Q were 6.48,14.18,24.56,and 29.86.The mean values of DNA were:5.64,1.76,1.24,and 0.80.The percentage of large DNA fragments averaged 56.08%,17.72%,12.68%,and 7.90%.Moreover,the Ct values of the internal control detected by PCR were 15.32,17.09,18.39,and 21.24.The three other groups exhibited significantly lower DNA concentration,higher N/Q ratios,decreased DNA quality and percentage of large fragments,and increased values of Ct,compared with the group of within 1 year of storage(P<0.05).The experimental results suggested that for novel ultrasound processed biopsy specimens,we should prioritize s

关 键 词：超声处理 存储年限 DNA质量 片段完整性 降解程度 二代测序 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R-331