不同来源的脱氧核糖核酸酶在基因表达量分析中应用效果的比较研究

Comparison of Application Effects of Different Kinds of DNase in Gene Expression Analysis

目的:比较在基因表达量分析中使用不同来源的脱氧核糖核酸酶(DNase)去除样本中残留DNA的应用效果差异,为研究者根据自己的实验需求选择合适的DNase提供依据。方法:选择国内外广泛使用的3种DNase,分别为Invitrogen DNase,TaKaRa DNase和Tiandz DNase。按照各种DNase的说明书对Trizol法提取的胎盘总RNA中残留DNA进行降解,定量方法采用实时荧光PCR测定反转录后的cDNA量。比较各种DNase对不同浓度RNA样本中残留DNA的处理效率、RNA损失量、操作时间及成本等。结果:三种DNase酶均能够完全降解不同残留量的DNA(10 ng、100 ng及1μg),实时荧光定量PCR均未检测到残留DNA。Invitrogen DNase对DNA的处理过程耗时最短,操作过程造成各种浓度RNA模板的损失量均最低,但成本最高;TaKaRa DNase处理的耗时居中,成本居中,RNA损失量最高,尤其对低浓度(<100 ng/μL)RNA样本;Tiandz DNase处理的耗时最长,成本最低,RNA损失量居中,当模板浓度较高(>500 ng/μL)时,损失量相对减少。结论:不同来源的DNase在基因表达量分析中对样本处理的应用效果各不相同,在不同样本分析中可参考以下原则正确使用合适的DNase,以保证分析的顺利和结果的准确:对于RNA提取量较多的样本,三种DNase均可选择,但如考虑成本,首选Tiandz DNase,如考虑耗时或RNA损失量,首选Invitrogen DNase,如既考虑耗时又考虑成本,可选TaKaRa DNase;对RNA提取量较少的血液或少量细胞(如单细胞)样本,应选择Invitrogen DNase。

Objective： To compare the application effect of different deoxyribonuclease （DNase） in gene expression analysis for providing the basis for the researchers to choose the suitable DNase according to their experimental requirements. Methods： Three kinds of commonly used DNase from domestic and foreign markets, including Invitrogen DNase, TaKaRa DNase and Tiandz DNase, were selected, The residual DNA in total RNA extracted from placenta by Trizol was degraded according to the instructions of DNase. Real-time PCR was performed for quantitative analysis of eDNA obtained from reverse transcription of RNA. DNA-degradation efficiency, RNA loss, operating time, and reagent cost of three kinds of DNase were compared. Results： All of three kinds of DNase can degrade DNA with the amounts of 10 ng, 100 ng and 1 I^g completely. There is no residual DNA which was detected by real-time PCR. lnvitrogen DNase had the shortest operation time, the least RNA loss in degrading RNA with various concentrations, but the highest cost. TaKaRa DNase had longer operation time, the most RNA loss （especially for RNA with low concentration, 〈100 ng/μL）, but the lower cost; Tiandz DNase had the longest operation time, the lowest cost and the relatively less RNA loss （especially for RNA with high concentration, 〉500 ng/μL）. Conclusion： The application effects of different kinds of DNase in gene expression analysis are different. The following basis can be used for selecting suitable DNase in the analysis of different samples to ensure the success of the analysis and accuracy of the results. For the tissue or the cell samples which contains much amount of RNA for analysis, all of three kinds of DNase can be chosen. However, if cost is considered, Tiandz DNase should be the first priority; if time or RNA loss is considered, lnvitrogen DNase would be the best choice; if both cost and time are considered, TaKaRa DNase can be chosen. For blood or a small amount of cells （such as single cell） which contains small amount ofRNA, Invitrogen DNase should be chosen.