Multiplex preamplification of specific cDNA targets prior to gene expression analysis by TaqMan Arrays.
Multiplex preamplification of specific cDNA targets prior to gene expression analysis by TaqMan Arrays.
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DOI：
10.1186/1756-0500-1-21
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发表时间：
2008-06-05
影响因子：
1.8
通讯作者：
Alcaraz, Antonio
中科院分区：
文献类型：
--
作者：
Mengual, Lourdes;Burset, Moises;Alcaraz, Antonio
背景：使用TaqMan阵列(TA)进行准确的基因表达定量可能会受到从一些临床样本中获得的RNA数量较少的限制。新型的cDNA预扩增系统，TaqMan Preamp Master Mix Kit(TaqMan Preamp Master Mix Kit，TaqMan Preamp Master Mix Kit)，能够多重预扩增靶基因，因此可以为其对TA的后继分析提供足够数量的特异扩增。FINDINGS：在TA分析之前，对22个样本进行了47个基因的多重预扩增，并比较了未预扩增(NPA)和预扩增(PA)样本的相对基因表达水平。总体而言，PA基因的平均周期阈值(CT)降低3.85(范围从2.07到5.01)。NPA和PA样本的基因表达测量值之间有很高的相关性(R)(平均r=0.970，范围从0.937到0.994；在所有选定的病例中p<；0.001)。在降解的RNA样本和/或低丰度表达基因的分析中，NPA和PA样本之间的相关性也很高。结论：在TA分析之前使用TPAMMK预扩增cDNA是一种可靠的方法，可以同时检测单个样本中多个靶点的基因表达。此外，这个过程在降解的RNA样本和低丰度表达的基因中得到了验证。这种结合的方法可能在临床研究中有广泛的应用，在临床研究中，通常获得的降解RNA数量很少，需要在每个样本中对几个基因进行量化。
BACKGROUND: An accurate gene expression quantification using TaqMan Arrays (TA) could be limited by the low RNA quantity obtained from some clinical samples. The novel cDNA preamplification system, the TaqMan PreAmp Master Mix kit (TPAMMK), enables a multiplex preamplification of cDNA targets and therefore, could provide a sufficient amount of specific amplicons for their posterior analysis on TA.FINDINGS: A multiplex preamplification of 47 genes was performed in 22 samples prior to their analysis by TA, and relative gene expression levels of non-preamplified (NPA) and preamplified (PA) samples were compared. Overall, the mean cycle threshold (CT) decrement in the PA genes was 3.85 (ranging from 2.07 to 5.01). A high correlation (r) between the gene expression measurements of NPA and PA samples was found (mean r = 0.970, ranging from 0.937 to 0.994; p < 0.001 in all selected cases). High correlation coefficients between NPA and PA samples were also obtained in the analysis of genes from degraded RNA samples and/or low abundance expressed genes.CONCLUSION: We demonstrate that cDNA preamplification using the TPAMMK before TA analysis is a reliable approach to simultaneously measure gene expression of multiple targets in a single sample. Moreover, this procedure was validated in genes from degraded RNA samples and low abundance expressed genes. This combined methodology could have wide applications in clinical research, where scarce amounts of degraded RNA are usually obtained and several genes need to be quantified in each sample.