Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.
Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.
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DOI：
10.1186/1756-0500-3-211
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发表时间：
2010-07-27
影响因子：
1.8
通讯作者：
Gonzalez-Escalona N
中科院分区：
文献类型：
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作者：
Rump LV;Asamoah B;Gonzalez-Escalona N
无DNA RNA的提取是逆转录PCR（RT-PCR）的关键步骤。每个RNA提取程序都会导致RNA样本被基因组DNA污染，这可能会在高灵敏度应用中导致假阳性结果，包括最近开发的定量实时PCR（RT-qPCR）检测，该检测针对invA mRNA用于检测活沙门氏菌细胞。这种特异性mRNA的测定可用于指示食物基质中肠道沙门氏菌的活细胞而不是死细胞的存在。我们评估了五种RNA提取试剂盒从指数生长的沙门氏菌细胞中制备RNA制剂的能力。根据回收的RNA总量、RNA分子的完整性和DNA的最小含量来判断用于下游应用（如RT-qPCR）的制剂的可接受性。这五种试剂盒产生的RNA制剂在产量、沙门氏菌RNA的完整性和污染物DNA的量方面存在显著差异。使用MasterPure试剂盒实现了最大的RNA回收率;然而，制备物含有高水平的基因组DNA。UltraClean提取试剂盒的RNA回收率较低，完整性较差。RNeasy Mini、RiboPure和PureLink提取试剂盒可产生高质量、不含DNA的RNA，适用于通过RT-qPCR检测沙门氏菌。我们发现RNeasy Mini和PureLink RNA提取试剂盒最适合通过RT-qPCR检测沙门氏菌invA mRNA。使用这两种试剂盒将大大降低假阳性结果的频率，并可能允许快速RT-qPCR测定食品样品中活沙门氏菌产生的invA mRNA。
The isolation of DNA-free RNA is a crucial step in the reverse transcription PCR (RT-PCR). Every RNA extraction procedure results in RNA samples contaminated with genomic DNA, which can cause false-positive outcomes in highly sensitive applications, including a recently developed quantitative real-time PCR (RT-qPCR) assay that targets invA mRNA for the detection of live Salmonella cells. The assay of this specific mRNA can be used to indicate the presence of live, as opposed to dead, cells of Salmonella enterica in a food matrix. We evaluated the ability of five RNA extraction kits to produce RNA preparations from exponentially growing Salmonella cells. The acceptability of the preparations for use in downstream applications such as RT-qPCR was judged in terms of the total amount of RNA recovered, the integrity of the RNA molecules, and minimal content of DNA. The five kits produced RNA preparations that differed markedly in yield, integrity of the Salmonella RNA and the amount of contaminant DNA. The greatest RNA recovery was achieved with the MasterPure kit; however, the preparation contained high levels of genomic DNA. The UltraClean extraction kit gave a low level of RNA recovery with a poor level of integrity. The RNeasy Mini, RiboPure and PureLink extraction kits produced high-quality, DNA-free RNA suitable for Salmonella detection by RT-qPCR. We showed that the RNeasy Mini and PureLink RNA extraction kits were the most suitable for the detection of Salmonella invA mRNA by RT-qPCR. The use of these two kits will greatly reduce the frequency of false-positive results and might allow fast RT-qPCR determination of invA mRNA produced by viable Salmonella in food samples.