Visualization of Gene Expression by Fluorescent Multiplex Reverse Transcriptase-PCR Amplification

Abstract

Many developmental and physiological analyses, population studies, and diagnostic tests can be performed by simply determining the presence or absence of a limited number of gene products. Here, we describe a rapid and sensitive procedure, based on the reverse transcription of total RNA samples followed by the co-amplification of specific complementary DNA molecules, for the simultaneous detection of different transcripts. Multiplex PCR amplification products are obtained in a single reaction mix containing several primer pairs, each of which includes a fluorescently labeled oligonucleotide; the amplification products are finally electrophoresed in an automated DNA sequencer controlled by fragment analysis software. The electropherograms obtained in this way allow a semiquantitative and efficient visualization of gene expression.