Clipsham R C, McCabe E R
Molecular Biology Institute, UCLA, Los Angeles, California 90095-1752, USA.
Mol Genet Metab. 2001 Dec;74(4):435-48. doi: 10.1006/mgme.2001.3261.
Molecular genetics is rapidly moving from simple identification of a gene of interest to characterization of gene products as components in complex networks. Critical tools for gene product analysis require a rapid method for evaluation of contextual expression. Here, we describe a robust, high primer density, single-tube, multiplex reverse transcription (HD-MRT) technique. This approach is capable of analyzing for the presence of numerous transcripts when polymerase chain reaction (PCR) is subsequently employed for individual gene-specific sequence amplification (HD-MRT-PCR). This assay substantially increases the total number of different cDNAs for amplification beyond previously published techniques. Our approach simultaneously eliminates RNA quality control issues for samples run in parallel while improving efficiency in the use of time and materials. This assay is designed for broad applicability and accessibility, employs modifications of commercially available components, and allows more than 25 independently selected gene-specific primers to be used simultaneously. Our protocol allows multiplexed primers to behave similarly to uniplex RT reactions, while avoiding potential interference between gene-specific and/or nonspecific primers during annealing and reverse transcription. Expression of putatively networked homologous transcripts was analyzed in multiple cell lines and tissues from mouse and human to validate the technique.
分子遗传学正迅速从简单鉴定感兴趣的基因转向将基因产物表征为复杂网络中的组成部分。基因产物分析的关键工具需要一种快速评估背景表达的方法。在此，我们描述了一种稳健的、高引物密度的单管多重逆转录（HD-MRT）技术。当随后使用聚合酶链反应（PCR）进行单个基因特异性序列扩增（HD-MRT-PCR）时，这种方法能够分析众多转录本的存在情况。该检测方法大大增加了可扩增的不同cDNA的总数，超过了先前发表的技术。我们的方法同时消除了平行运行样本的RNA质量控制问题，同时提高了时间和材料的使用效率。该检测方法旨在具有广泛的适用性和可及性，采用了对市售组件的改进，并允许同时使用超过25种独立选择的基因特异性引物。我们的方案使多重引物的行为类似于单重RT反应，同时避免了退火和逆转录过程中基因特异性和/或非特异性引物之间的潜在干扰。在来自小鼠和人类的多种细胞系和组织中分析了假定的网络同源转录本的表达，以验证该技术。
