Differential gene expression profiles are dependent upon method of peripheral blood collection and RNA isolation.
Differential gene expression profiles are dependent upon method of peripheral blood collection and RNA isolation.
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差异基因表达谱取决于外周血收集和RNA分离的方法。
DOI：
10.1186/1471-2164-9-474
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发表时间：
2008-10-10
期刊：
影响因子：
4.4
通讯作者：
Seyfert-Margolis, Vicki
中科院分区：
文献类型：
--
作者：
Asare, Adam L.;Kolchinsky, Svetlana A.;Gao, Zhong;Wang, Richard;Raddassi, Khadir;Bourcier, Katarzyna;Seyfert-Margolis, Vicki
RNA的分离和纯化步骤对基因表达谱的结果影响很大。有两种市售产品用于全血RNA采集，PAXgene™和Tempus™血液采集管，每个都有自己的RNA纯化方法。在这两种系统中，血液被收集到试管中后立即被溶解，并使用专有试剂稳定RNA。这两种系统使血液处理程序最小化，从而最大限度地减少通过血液处理或处理诱导基因表达变化的风险。由于RNA纯化步骤可能会影响总RNA池，因此我们使用PAXgene™或Tempus™方法检测了RNA分离对基因表达谱的影响。使用微阵列作为受刺激全血RNA的读出器，我们在PAXgene™或Tempus™的RNA样本中发现了一组共同的表达转录物。然而，我们也发现了一些依赖于收集管类型的独特表达，这表明RNA纯化方法影响差异基因表达谱的结果。具体来说，当使用ABI Tempus™方法分离RNA时，在受刺激样品中发现了几种已知的pha诱导基因的转录本，包括IFNγ、IL13、IL2、IL3和IL4，而使用PAXgene™方法则没有（p < 0.01， FDR校正）。Sequenom quantitative Gene Expression (QGE) （SanDiego， CA）检测证实，来自PHA刺激细胞的Tempus™纯化RNA中IL2、IL4和IFNγ上调，而使用PAXgene™纯化的RNA中只有IL2上调（p < 0.05）。在这里，我们证明了外周血RNA分离方法可以严重影响差异表达结果，特别是在临床环境中，fold-change差异通常很小，并且在生物队列中存在固有的可变性。研究发现，基于Tempus™系统的改进方法产量高，杂交后阵列质量好，表达量变化小，并且产生的差异表达结果与预测的PHA刺激免疫效应一致。
RNA isolation and purification steps greatly influence the results of gene expression profiling. There are two commercially available products for whole blood RNA collection, PAXgene™ and Tempus™ blood collection tubes, and each comes with their own RNA purification method. In both systems the blood is immediately lysed when collected into the tube and RNA stabilized using proprietary reagents. Both systems enable minimal blood handling procedures thus minimizing the risk of inducing changes in gene expression through blood handling or processing. Because the RNA purification steps could influence the total RNA pool, we examined the impact of RNA isolation, using the PAXgene™ or Tempus™ method, on gene expression profiles. Using microarrays as readout of RNA from stimulated whole blood we found a common set of expressed transcripts in RNA samples from either PAXgene™ or Tempus™. However, we also found several to be uniquely expressed depending on the type of collection tube, suggesting that RNA purification methods impact results of differential gene expression profiling. Specifically, transcripts for several known PHA-inducible genes, including IFNγ, IL13, IL2, IL3, and IL4 were found to be upregulated in stimulated vs. control samples when RNA was isolated using the ABI Tempus™ method, but not using the PAXgene™ method (p < 0.01, FDR corrected). Sequenom Quantiative Gene Expression (QGE) (SanDiego, CA) measures confirmed IL2, IL4 and IFNγ up-regulation in Tempus™ purified RNA from PHA stimulated cells while only IL2 was up-regulated using PAXgene™ purified (p < 0.05). Here, we demonstrate that peripheral blood RNA isolation methods can critically impact differential expression results, particularly in the clinical setting where fold-change differences are typically small and there is inherent variability within biological cohorts. A modified method based upon the Tempus™ system was found to provide high yield, good post-hybridization array quality, low variability in expression measures and was shown to produce differential expression results consistent with the predicted immunologic effects of PHA stimulation.
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影响因子：
12.3
作者：
Gentleman RC;Carey VJ;Bates DM;Bolstad B;Dettling M;Dudoit S;Ellis B;Gautier L;Ge Y;Gentry J;Hornik K;Hothorn T;Huber W;Iacus S;Irizarry R;Leisch F;Li C;Maechler M;Rossini AJ;Sawitzki G;Smith C;Smyth G;Tierney L;Yang JY;Zhang J
通讯作者：
Zhang J
影响因子：
5.3
作者：
Thomson, SAM;Wallace, MR
通讯作者：
Wallace, MR
影响因子：
3.7
作者：
Abdalla, AO;Kiaii, S;Rabbani, H
通讯作者：
Rabbani, H
DOI：
10.1073/pnas.0630494100
发表时间：
2003-03-18
影响因子：
11.1
作者：
Ding, CM;Cantor, CR
通讯作者：
Cantor, CR
影响因子：
2.7
作者：
Saeed, AI;Sharov, V;Quackenbush, J
通讯作者：
Quackenbush, J