Vermeulen Joëlle, Derveaux Stefaan, Lefever Steve, De Smet Els, De Preter Katleen, Yigit Nurten, De Paepe Anne, Pattyn Filip, Speleman Frank, Vandesompele Jo
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
BMC Res Notes. 2009 Nov 25;2:235. doi: 10.1186/1756-0500-2-235.
The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR.
The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification.
Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.
定量聚合酶链反应（qPCR）是一种广泛用于基因表达分析的方法。然而，材料不足常常会影响大规模基因表达研究。本研究的目的是评估一种RNA预扩增方法，以产生微克级的cDNA作为qPCR的输入。
首先通过测量六个神经母细胞瘤细胞系RNA样本中20个基因以及两个市售参考RNA样本中194个基因在预扩增前后的表达，对线性等温Ribo-SPIA预扩增方法（WT-Ovation；NuGEN）进行评估，随后将其应用于从神经母细胞瘤肿瘤中提取的738个RNA样本的大样本组。对所有RNA样本进行RNA完整性和纯度评估。从5至50纳克总RNA开始应用样本预扩增方法，产生约5微克的cDNA，足以检测1000多个靶基因。本研究获得的结果表明，预扩增cDNA的产量恒定，与输入RNA的量无关；预扩增后差异基因表达得以保留，且未引入实质性偏差；未共扩增污染的基因组DNA；无需纯化预扩增材料；最后表明了良好RNA质量对于实现预扩增的重要性。
应用这种无偏差且易于使用的样本预扩增技术为生成足够的材料用于诊断和预后检查提供了巨大优势，并能够使用有限量的样本材料进行大规模qPCR基因表达研究。
