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

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. | Turkish Journal of Biology Research Article Turk J Biol (2015) 39: 567-574 © TÜBİTAK doi: Optimization of multiplex RT-PCR for M1, M23, and M23X splice variants of AQP4 and β-actin transcripts in Dalton’s lymphoma mouse tissues Rajaneesh Kumar GUPTA, Sukala PRASAD* Molecular Biology and Biochemistry Lab, Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi, Uttar Pradesh, India Received: Accepted/Published Online: Printed: Abstract: 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, , , and , 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. β-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. Key words: MRT-PCR, AQP4, Dalton’s lymphoma, gene expression, primer .