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Multiplex reverse transcriptase-polymerase chain reaction (MRT-PCR) is a method of choice for efficient and simultaneous analysis of gene expression at the transcript level with limited amounts of tissues; however, it is less common than other methods due to certain limitations. Here, we describe the protocol for MRT-PCR-based semiquantitative analysis of all AQP4 transcripts simultaneously in Dalton's lymphoma (DL) using transcript-specific primers and outline the critical parameters. Aquaporin-4 (AQP4) water channels are associated with edema of tissues due to various diseases. The AQP4 transcript possesses alternative transcription initiation sites and produces three isoforms, AQP4.M1, AQP4.M23, and AQP4.M23X, which constitute heterotetrameric orthogonal arrays of particles (OAPs) in the plasma membrane. In order to validate the MRT-PCR technique, the AQP4 transcripts were also analyzed in various tissues of mice with DL. A-Actin transcript was simultaneously amplified as a reference to monitor the possible intra- and interassay variations during MRT-PCR reactions and for relative quantification of target mRNAs. Our data suggest that the MRT-PCR technique is suitable for simultaneous detection and semiquantitative analysis of AQP4 transcripts, and can also be used as a routine tool for simultaneous analysis of multiple transcripts in other tissues.

Optimization of multiplex RT-PCR for M1, M23, and M23X splice variantsof AQP4 and β-actin transcripts in Dalton’s lymphoma mouse tissues