Spandidos Athanasia, Wang Xiaowei, Wang Huajun, Dragnev Stefan, Thurber Tara, Seed Brian
Center for Computational and Integrative Biology, Massachusetts General Hospital, MA, USA.
BMC Genomics. 2008 Dec 24;9:633. doi: 10.1186/1471-2164-9-633.
Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile.
We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method.
We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays.
定量聚合酶链反应（QPCR）是一种广泛应用的用于准确测定转录本丰度的分析方法。用于QPCR的引物已在基因组规模上进行设计，但非靶基因的非特异性扩增一直是个问题。尽管已经创建了几个在线数据库用于存储和检索经过实验验证的引物，但现有数据库中目前仅存在几千对引物，并且这些引物并非设计用于常见的PCR热循环条件。
我们之前报道了一种算法的实施，用于预测大多数已知人类和小鼠基因的PCR引物。我们现在报告利用该资源鉴定出17483对已通过实验验证可扩增对应于不同小鼠转录本的独特序列的引物。这些引物对已通过凝胶电泳、DNA序列分析和热变性曲线进行了验证。除了验证研究外，我们还使用这些引物确定了扩增的均匀性以及使用流行且廉价的SYBR Green I检测方法进行QPCR反应的技术可重复性。
我们已经鉴定出一组经过实验验证的用于PCR和QPCR的小鼠引物对，它们可在常见的PCR热循环条件下使用，从而能够并行评估大量基因的转录本丰度。这一特性对于确认和/或更精确地呈现从DNA微阵列实验中观察到的数据趋势越来越有吸引力。
