Werbrouck Hadewig, Botteldoorn Nadine, Uyttendaele Mieke, Herman Lieve, Van Coillie Els
Flemish Government, Institute for Agricultural and Fisheries Research, Unit Technology and Food, Brusselsesteenweg 370, 9090 Melle, Belgium.
J Microbiol Methods. 2007 May;69(2):306-14. doi: 10.1016/j.mimet.2007.01.017. Epub 2007 Feb 7.
In the current study, various steps in the real-time reverse transcription PCR (real-time RT-PCR) method for determination of RNA expression levels starting from different numbers of Listeria monocytogenes cells were evaluated and optimized. Our results showed that the RNA isolation method as well as the cDNA synthesis may influence the sensitivity of the procedure. For high bacterial cell numbers (10(9) bacterial cells), the RNAqueous kit and the RNeasy Mini kit were equally useful, whereas for low bacterial cell numbers (<or=10(7) bacterial cells) the RNAqueous-Micro kit was found to be the most sensitive RNA isolation kit. For cDNA synthesis, the use of random hexamers and an incubation time of 90 min with the Multiscribe RT-enzyme resulted in the highest efficiency of conversion of RNA into cDNA. To compare RNA levels of different L. monocytogenes strains, it is necessary to analyse the expression levels at the same point in the growth phase and to have a 100% matching of the primers for all tested strains to obtain reliable results. In general, our results showed that real-time RT-PCR needs to be optimized to obtain reliable and accurate data and that many factors can influence the outcome of the real-time RT-PCR data.
在本研究中，对从不同数量的单核细胞增生李斯特菌细胞开始测定RNA表达水平的实时逆转录PCR（实时RT-PCR）方法中的各个步骤进行了评估和优化。我们的结果表明，RNA分离方法以及cDNA合成可能会影响该程序的灵敏度。对于高细菌细胞数（10⁹个细菌细胞），RNAqueous试剂盒和RNeasy Mini试剂盒同样有用，而对于低细菌细胞数（≤10⁷个细菌细胞），发现RNAqueous-Micro试剂盒是最灵敏的RNA分离试剂盒。对于cDNA合成，使用随机六聚体并与Multiscribe RT酶孵育90分钟可使RNA转化为cDNA的效率最高。为了比较不同单核细胞增生李斯特菌菌株的RNA水平，有必要在生长阶段的同一点分析表达水平，并使所有测试菌株的引物100%匹配以获得可靠的结果。总体而言，我们的结果表明，实时RT-PCR需要进行优化以获得可靠和准确的数据，并且许多因素会影响实时RT-PCR数据的结果。
