分离白细胞法、低渗红细胞法、全血直提法提取的冻存血RNA质量及其反转录后基因扩增效果

Quality of frozen blood RNA extracted by separating peripheral blood leukocyte method,hypotonic red cell method and whole blood direct extraction method and its gene amplification effect after reverse transcription

目的观察分离白细胞法、低渗红细胞法、全血直提法提取的冻存血RNA质量及其反转录后基因扩增效果。方法将冻存血标本12例份随机分为3组,分别采用分离白细胞法、低渗红细胞法、全血直提法提取RNA,提取出的RNA溶液分别记为A、B、C组。使用NANODROP 1000分光光度计测算各组RNA浓度及OD(A260/A280)值、OD(A260/A230)值,以OD(A260/A280)值、OD(A260/A230)值表示RNA纯度。采用琼脂糖凝胶电泳法观察各组RNA完整性。各组RNA反转录后,采用PCR法扩增,观察扩增产物电泳条带情况;各组RNA反转录后,采用qPCR法扩增,计算循环阈值(Ct值)。结果A组RNA浓度、OD(A260/A280)值、OD(A260/A230)值均高于B、C组(P均<0.05)。A组琼脂糖凝胶电泳没有出现任何条带,B组条带严重弥散,C组有条带存在。C组RNA反转录后得到的扩增产物条带最为明亮。A、B、C组Ct值分别为28.27±1.01、27.49±0.22、24.20±0.25,其中C组Ct值与A、B组相比,P均<0.05。结论与分离白细胞法、低渗红细胞法相比,全血直提法提取的冻存血RNA完整性更好,其反转录后基因扩增效果更高。

Objective To observe the quality of RNA extracted from frozen blood by separating peripheral blood leukocyte,hypotonic red cell and whole blood direct extraction method,respectively and its gene amplification after reverse transcription.Methods Twelve cases of frozen blood were randomly divided into 3 groups.We used the method of peripheral blood leukocyte,hypotonic red cell and whole blood direct extraction to extract RNA,and the extracted RNA solution was recorded as group A,group B,and group C,respectively.RNA concentration,OD value(A260/A280),and OD value(A260/A230)were measured by nanodrop 1000 spectrophotometer.OD value(A260/A280)and OD value(A260/A230)were used to express the purity of RNA.The integrity of each group of RNA was observed by the agarose gel electrophoresis method.After the reverse transcription of RNA in each group,the PCR method amplification was used to observe the electrophoresis strip of the augmentation material,and the qPCR method was used to amplification and the cycle threshold(Ct value)was calculated.Results The RNA concentration,OD value(A260/A280)and OD value(A260/A230)of group A were higher than those of groups B and C(all P<0.05).No band appeared in the agarose gel electrophoresis of group A,bands were severely dispersed in the group B,and bands existed in the group C.The band of amplification resulting from the reverse transcription of group C was the brightest,Ct values in the groups A,B,and C were 28.27±1.01,27.49±0.22,and 24.20±0.25 respectively,and significant difference was found in the Ct between the group C and groups A and B(all P<0.05).Conclusion The frozen blood RNA extracted by whole blood direct extraction method has better integrity and higher gene amplification effect after reverse transcription as compared with the methods of separating peripheral blood leukocyte and hypotonic red cell.