Arvidsson Samuel, Kwasniewski Miroslaw, Riaño-Pachón Diego Mauricio, Mueller-Roeber Bernd
Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany.
BMC Bioinformatics. 2008 Nov 1;9:465. doi: 10.1186/1471-2105-9-465.
Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays.
Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet http://www.quantprime.de/ or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96%.
QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays.
在基因组学研究中，使用定量聚合酶链反应（qPCR）分析进行中大规模表达谱分析变得越来越重要。实验准备中的一个主要瓶颈是特异性引物对的设计，研究人员必须做出几个明智的选择，而这些选择往往超出了他们的专业领域。使用现有的引物设计工具，必须做出几个交互式决策，导致设计过程冗长，且分析质量参差不齐。
在此，我们展示了QuantPrime，这是一种直观、用户友好的全自动工具，用于从小规模到大规模qPCR分析的引物对设计。QuantPrime可以通过互联网http://www.quantprime.de/在线使用，也可以在下载后在本地计算机上使用；它提供具有高度可定制参数的设计和特异性检查，并可与许多重要高等真核模式生物和植物作物的公开转录组（目前总共295种）一起使用，同时受益于可用基因组注释中的外显子-内含子边界和可变剪接变体信息。对模式植物拟南芥、作物大麦和模式绿藻莱茵衣藻的实验结果表明，设计的引物对成功率超过96%。
如实验数据所示，QuantPrime构成了一个灵活的全自动网络应用程序，可用于在更大规模的qPCR实验中进行可靠的引物设计。这个灵活的框架也易于在其他定量应用中使用，如qPCR的水解探针设计和定量原位杂交的寡核苷酸探针设计。用户提出的未来建议可以很容易地实现，从而使QuantPrime能够发展成为一个用于RNA表达分析设计的广泛平台。
