不同保存和处理方式对全血标本总RNA提取的影响  

Effects of different preservation condition and processing methods on RNA isolation from whole blood

作　　者：何松哲 何慧[1,2] 陈懿[1,2] 陈羽[3] 余道军[1,2] 

机构地区：[1]浙江中医药大学附属杭州第一医院,浙江杭州310006 [2]杭州市第一人民医院,浙江杭州310006 [3]温州医科大学,浙江温州325035

出　　处：《中国微生态学杂志》2015年第2期224-226,共3页Chinese Journal of Microecology

基　　金：杭州市科技局社会发展科研计划资助项目(20130733Q04);杭州市卫生科技计划重大项目(2012ZD001)

摘　　要：目的分析全血标本RNA提取的影响因素,并对全血RNA提取条件进行优化。方法收集100例全血标本,混匀后分为低渗红细胞溶解组(A组,50例)和全血直接提取组(B组,50例),利用Trizol试剂提取放置在不同温度、不同保存时间的全血标本总RNA,并采用Ur-2401紫外分光光度计测定其浓度和纯度;另选60例全血标本作为反复冻融组(C组),取不同次数反复冻融的全血标本利用Trizol法直接提取其RNA。结果 A组和B组新鲜标本中所获取的RNA浓度分别为450.0 ng/μL和458.8 ng/μL,37℃保存7 d标本中的RNA浓度分别为374.8 ng/μL和375.2 ng/μL,不同的保存温度与时间均对总RNA浓度(P=0.003、P=0.002)和纯度(P值均为0.011)的差异均有统计学意义,而相同条件下提取的总RNA浓度和纯度的差异无统计学意义(P=0.984、P=0.311);C组全血标本反复冻融的次数与保存时间对总RNA浓度(P=0.002、P=0.001)、纯度(P值均为0.008)差异均有统计学意义,而保存温度对总RNA浓度和纯度差异无统计学意义(P=0.611、P=0.362)。结论不同条件下全血标本中所获取的总RNA浓度有所差异,但在总RNA纯度和成功率方面,低渗破坏红细胞法优于直接全血提取法,反复冻融影响全血RNA提取效率和得率。Objective To explore the influencing factors in RNA extraction from blood samples and optimize the method of RNA isolation. Methods 160 blood samples were collected and divided into three groups： low permea- bility group （ group A, n = 50） , direct RNA extraction group （ group B, n = 50 ） and repeated freeze-thaw group （ group C, n = 60）. Total RNA was extracted by Trizol reagent from the samples in group A and B stored in different temperatures and durations, and from the samples in group C with repeated freeze-thaw method. The concentration and purity were measured using Ur-2401 Ultraviolet Spectrophotometer. Results The concentrations of RNA from fresh samples of group A and B were 450. 0 ng/p.L and 458.8 ng/μL respectively; when the samples were stored at 37 ℃ for 7 days, the RNA concentrations were 374. 8 ng/μL and 375.2 ng/μL respectively. The preservation temperature and period had an impact on the total RNA concentration （P = 0. 003, P = 0. 002） and purity （P =0. 011, P =0. 011 ） in both group A and B with statistical significance, while the differences were not statistically significant in the concentration and purity （P = 0. 984 and P = 0. 311 ） of total RNA extracted under the same condition. In group C, both the repeated freeze-thaw cycles and preservation period had a statistically significant impact on total RNA concentration （ P = 0. 002 and P = 0. 001 ） and purity （ P = 0. 008 ）, while the preservation temperature didn＇t （P = 0. 611 and P = 0. 362）. Conclusion The total RNA concentrations are different when extracted under different conditions. In terms of purity and yield of RNA, the low permeability method is slightly bet- ter than direct RNA extraction from whole blood, and the repeated freeze-thaw method will affect the productivity of whole blood RNA extraction.

关 键 词：外周全血 RNA提取 RNA浓度 RNA纯度 

分 类 号：R44[医药卫生—诊断学] R53[医药卫生—临床医学]