Real-time reverse transcriptase polymerase chain reaction: an improvement in detecting mRNA levels in mouse cranial tissue.
Real-time reverse transcriptase polymerase chain reaction: an improvement in detecting mRNA levels in mouse cranial tissue.
复制标题
实时逆转录酶聚合酶链式反应：检测小鼠颅组织中 mRNA 水平的改进。
DOI：
10.1097/01.prs.0000219339.82624.b9
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发表时间：
2006
影响因子：
3.6
通讯作者：
Gosain,ArunK
中科院分区：
文献类型：
--
作者：
Singh,Rashmi;Recinos,RenéF;Agresti,Michael;Schaefer,RichardB;Bosbous,Mark;Gosain,ArunK
背景：信使RNA水平的定量传统上通过北方印迹分析进行。虽然这被认为是标准方法，但它很耗时，需要大量的RNA。逆转录聚合酶链反应是一种半定量方法，已被用作北方印迹法的更快速和更灵敏的替代方法。实时逆转录酶聚合酶链反应是一种定量技术，作为一种快速可靠的mRNA定量方法而获得广泛认可。由于这两种技术目前正在使用的小鼠颅缝模型中的基因表达进行评估，本研究进行了比较的灵敏度和变化的实时到传统的逆转录酶聚合酶链反应在这个model.Methods：小鼠脑RNA的分离和扩增使用实时和常规的方法。对于实时方法，进行RNA的连续10倍稀释，范围为1 fg至100 ng。对于常规方法，确定了一致聚合酶链反应扩增所需的最小RNA量。转化生长因子β-1和β-肌动蛋白RNA转录测定使用这两种techniques.Results：一个毫微微克的RNA可以检测到的实时方法，虽然需要10 fg可靠地检测差异; 500 ng的RNA需要一致的聚合酶链反应扩增使用传统的方法。实时逆转录聚合酶链反应的变异性时，表示为变异系数（SD作为平均值的百分比）范围从0.23至2.6%的所有基因测试，相比之下，9至70%的常规逆转录聚合酶链反应。结论：实时逆转录聚合酶链反应被成功地用于检测mRNA从不同的小鼠基因。实时方法在检测少量mRNA方面比北方印迹分析和常规聚合酶链反应灵敏得多。对于所有测试的基因，实时方法的变异性比作者实验室中进行的常规方法低10倍以上。
Background:Quantitation of messenger RNA levels has traditionally been carried out by Northern blot analysis. While this is regarded as the standard method, it is time-consuming and requires large quantities of RNA. Reverse-transcriptase polymerase chain reaction is a semiquantitative method that has been used as a more rapid and sensitive alternative to Northern blotting. Real-time reverse-transcriptase polymerase chain reaction is a quantitative technique that is gaining widespread acceptance as a rapid and reliable way of quantifying mRNA. Since both techniques are currently being used to evaluate gene expression in the murine cranial suture model, the present study was performed to compare the sensitivity and variability of real-time to conventional reverse-transcriptase polymerase chain reaction in this model.Methods:Mouse brain RNA was isolated and amplified using real-time and conventional methods. For the real-time method, a serial 10-fold dilution of RNA, ranging from 1 fg to 100 ng, was performed. For the conventional method, the minimum amount of RNA needed for consistent polymerase chain reaction amplification was determined. Transforming growth factor beta-1 and β-actin RNA transcripts were measured using both techniques.Results:One femtogram of RNA could be detected by the real-time method, although 10 fg were required to reliably detect differences; 500 ng of RNA was required for consistent polymerase chain reaction amplification using the conventional method. The variability of real-time reverse-transcriptase polymerase chain reaction when expressed as a coefficient of variation (SD as a percentage of the mean) ranged from 0.23 to 2.6 percent for all genes tested, as compared with 9 to 70 percent for conventional reverse-transcriptase polymerase chain reaction.Conclusions:Real-time reverse-transcriptase polymerase chain reaction was used successfully to detect mRNA from different mouse genes. The real-time method is much more sensitive in detecting small amounts of mRNA than both Northern blot analysis and conventional polymerase chain reaction. The variability of the real-time method is more than 10-fold lower compared with the conventional method performed in the authors’ laboratory for all genes tested.