Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods.
Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods.
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测量 microRNA：微阵列和定量 PCR 测量以及不同总 RNA 制备方法的比较。
DOI：
10.1186/1472-6750-8-69
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发表时间：
2008-09-11
影响因子：
3.5
通讯作者：
Curry, Bo
中科院分区：
文献类型：
--
作者：
Ach, Robert A.;Wang, Hui;Curry, Bo
鉴于microRNAs(MiRNAs)在细胞调控中的重要作用，确定这些分子的表达水平在生物学的许多领域引起了研究人员的极大兴趣。测量总RNA样本中miRNAs的两种常见方法是微阵列和定量RT-PCR(QPCR)。为了理解使用这两种不同技术来测量miRNAs的研究结果，了解这两种分析方法的结果之间的相关性是很重要的。由于这两种方法都使用总RNA作为起始材料，因此了解所使用的总RNA制备的特定方法可能如何影响miRNAs的测量也是至关重要的。我们使用安捷伦微阵列测量了470个人类miRNAs在9个人类组织中的表达，并将这些结果与相同组织中61个miRNAs的qPCR图谱进行了比较。大多数表达的miRNAs(53/60)在两种方法之间有很好的相关性(R>；0.9)。使用添加合成的miRNAs，我们进一步检查了相关性最低的两个miRNAs，发现差异不能归因于两种方法的差异敏感性。我们还使用miRNA微阵列测试了三种广泛使用的总RNA样本制备方法。我们发现，虽然几乎所有的miRNA水平在这三种方法之间都是一致的，但当通过微阵列分析测量时，有一些miRNA的水平在不同的prep技术之间始终存在差异。这些差异得到了qPCR测量的证实。Agilent miRNA微阵列结果和qPCR结果之间的相关性以及不同总RNA制备方法之间的相关性一般都很好。然而，有一些miRNAs的水平在微阵列和qPCR测量之间，或者在不同的样品制备方法之间没有相关性。因此，研究人员在比较使用不同分析或样品制备方法获得的结果时应谨慎行事。
Determining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be affected by the particular method of total RNA preparation used. We measured the expression of 470 human miRNAs in nine human tissues using Agilent microarrays, and compared these results to qPCR profiles of 61 miRNAs in the same tissues. Most expressed miRNAs (53/60) correlated well (R > 0.9) between the two methods. Using spiked-in synthetic miRNAs, we further examined the two miRNAs with the lowest correlations, and found the differences cannot be attributed to differential sensitivity of the two methods. We also tested three widely-used total RNA sample prep methods using miRNA microarrays. We found that while almost all miRNA levels correspond between the three methods, there were a few miRNAs whose levels consistently differed between the different prep techniques when measured by microarray analysis. These differences were corroborated by qPCR measurements. The correlations between Agilent miRNA microarray results and qPCR results are generally excellent, as are the correlations between different total RNA prep methods. However, there are a few miRNAs whose levels do not correlate between the microarray and qPCR measurements, or between different sample prep methods. Researchers should therefore take care when comparing results obtained using different analysis or sample preparation methods.
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影响因子：
14.9
作者：
Imbeaud S;Graudens E;Boulanger V;Barlet X;Zaborski P;Eveno E;Mueller O;Schroeder A;Auffray C
通讯作者：
Auffray C
影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
影响因子：
14.9
作者：
Griffiths-Jones, S
通讯作者：
Griffiths-Jones, S
影响因子：
30.8
作者：
Bentwich, I;Avniel, A;Bentwich, Z
通讯作者：
Bentwich, Z
影响因子：
64.5
作者：
Eulalio, Ana;Huntzinger, Eric;Izaurralde, Elisa
通讯作者：
Izaurralde, Elisa