Single-tube gene-specific expression analysis by high primer density multiplex reverse transcription

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.