Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays
Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays
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DOI：
10.1016/j.jim.2003.10.004
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发表时间：
2003-12-01
影响因子：
2.2
通讯作者：
Stenger, DA
中科院分区：
文献类型：
--
作者：
Thach, DC;Lin, BC;Stenger, DA
在流行病学监测的背景下，人类血液样本的全基因组表达研究面临着许多挑战，其中最重要的是能够产生可靠的检测转录水平。这促使我们考虑Paxgene（TM）血液RNA系统，它由真空采血管（PAX管）中的稳定添加剂和样品处理试剂盒（PAX试剂盒）组成。PAX管含有一种溶液，当血液被吸入管中时，该溶液抑制RNA降解和基因诱导。在实际临床应用的条件下，PAX管中RNA的稳定性已经通过RT-PCR（Clin.Chem.48（2002）1883）确定，但尚未在微阵列上在转录组水平上进行评估。在这里，我们报告了一个质量保证和控制的协议，能够产生可靠的基因表达谱使用基因芯片（R）系统与RNA分离PAX管。使用该方案，我们比较了RNA的质量指标和基因表达谱，从血液中提取的PAX管，坐在室温下2小时，与血液中的PAX管在室温下孵育9小时，然后在-20 ℃储存6天。在众多指标中，检测到两种处理方法之间的DNA污染水平、RNA产率和双链cDNA产率的差异。基因表达的方差分析显示处理方法之间存在微小但显著的差异。这些结果有助于确定临床研究的方案，并使我们朝着在诊断和监测中使用转录组的目标前进。(C)2003 Elsevier B. V.保留所有权利。
Genome-wide expression studies of human blood samples in the context of epidemiologic surveillance are confronted by numerous challenges-one of the foremost being the capability to produce reliable detection of transcript levels. This led us to consider the Paxgene(TM) Blood RNA System, which consists of a stabilizing additive in an evacuated blood collection tube (PAX tube) and a sample processing kit (PAX kit). The PAX tube contains a solution that inhibits RNA degradation and gene induction as blood is drawn into the tube. The stability of RNA in PAX tubes under conditions for practical clinical applications has been determined by RT-PCR (Clin. Chem. 48 (2002) 1883), but has not been assessed at the transcriptome level on Affymetrix(R) microarrays. Here, we report a quality assured and controlled protocol that is capable of producing reliable gene expression profiles using the GeneChip(R) system with RNA isolated from PAX tubes. Using this protocol, we compared quality metrics and gene-expression profiles of RNA, extracted from blood in PAX tubes that sat at room temperature for 2 h, with that of blood in PAX tubes incubated at room temperature for 9 h followed by storage at - 20 degreesC for 6 days. Of numerous metrics, differences between the two handling methods were detected for the level of DNA contamination, RNA yield, and double stranded cDNA yield. Analysis of variance of gene-expression revealed small but significant differences between the handling methods. These results contribute to the determination of protocols for clinical studies and progress us towards the goal of using the transcriptome in diagnosis and surveillance. (C) 2003 Elsevier B.V. All rights reserved.