Recovery of microarray-quality RNA from frozen EDTA blood samples
Recovery of microarray-quality RNA from frozen EDTA blood samples
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DOI：
10.1016/j.vascn.2008.10.003
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发表时间：
2009-01-01
影响因子：
1.9
通讯作者：
Heubach, Juergen F.
中科院分区：
文献类型：
--
作者：
Beekman, Johanna M.;Reischl, Joachim;Heubach, Juergen F.
简介：具有RNA稳定添加剂（例如PAXgene）的血液采集系统的可用性为大型多中心临床试验中的基因表达谱分析开辟了领域。在这里，我们研究了PAXgene系统是否也提供了一种从冷冻EDTA血液样品中提取RNA的方法，当用标准方案提取时，这种方法不能产生足够高质量的RNA用于RNA表达谱分析。方法：从6名健康志愿者中采集全血，置于常规EDTA管中并冷冻。将解冻的EDTA血液转移至PAXgene管中，并使用PAXgene RNA提取试剂盒提取RNA。进行微阵列分析以评估RNA质量对基因表达谱的影响。结果：转移样品的RNA产量为1.76 +/- 0.88 μ g/ml。该产率明显低于PAXgene参照组的产率（2.84 +/-0.62 μ g/ml），但显著高于通常应用于新鲜EDTA血样的标准方案的产率（0.07 +/-0.06 μ g/ml）。转移样品的RNA完整性数值（RIN）为6.1 +/- 0.8，而PAXgene参比品为9.8 +/- 0.1。提取的RNA的微阵列分析表明，RIN值高于5的样品产生的数据满足制造商定义的质量标准。讨论内容：将解冻的EDTA血液转移到PAXgene血液收集管中提供了一种方法来回收足够的可接受质量的RNA用于微阵列实验。(C)2008年爱思唯尔公司All rights reserved.
Introduction: The availability of blood collection systems with RNA stabilizing additives (e.g. PAXgene) has opened the field for gene expression profiling in large multi-center clinical trials. Here, we investigated whether the PAXgene system also offers a method for extraction of RNA from frozen EDTA blood samples, which do not yield RNA of high enough quality for RNA expression profiling, when extracted with standard protocols. Methods: Whole blood was obtained from six healthy volunteers in conventional EDTA tubes and frozen. The thawed EDTA blood was transferred into PAXgene tubes, and the RNA was extracted using the PAXgene RNA extraction kit. Microarray analysis was performed to asses the effect of RNA quality on gene expression profiles. Results: The RNA yield of the transferred samples was 1.76 +/- 0.88 mu g/ml. This yield was clearly lower than the yield from a PAXgene reference group (2.84 +/- 0.62 mu g/ml), but considerably higher than the yield resulting from a standard protocol usually applied to fresh EDTA blood samples (0.07 +/- 0.06 mu g/ml). The RNA integrity number (RIN) of the transferred samples was 6.1 +/- 0.8 as compared to 9.8 +/- 0.1 for the PAXgene reference. Microarray analysis of the extracted RNA suggested that samples with RIN values above 5 produce data that fulfill the quality criteria defined by the manufacturer. Discussion: The transfer of thawed EDTA blood into PAXgene blood collection tubes offers a method to recover sufficient RNA of acceptable quality for microarray experiments. (C) 2008 Elsevier Inc. All rights reserved.