Development of an isoform-discriminating RT-qPCR assay for transcript-level profiling of human ASAH2 in colorectal cancer cell models.

N-Acylsphingosine Amidohydrolase 2 (ASAH2) encodes neutral ceramidase (nCDase), one of several ceramidases involved in sphingolipid metabolism. nCDase hydrolyzes ceramide into sphingosine and contributes to the regulation of the ceramide-sphingosine-1-phosphate signaling axis. Compared with acid and alkaline ceramidases, nCDase remains less well characterized, and the expression and functional relevance of alternatively spliced ASAH2 isoforms are poorly understood. Dysregulated ASAH2 expression has been implicated in colorectal cancer (CRC), suggesting that transcript-level analysis may provide useful information regarding ASAH2 regulation in tumor-related contexts. Human ASAH2 exists as two alternatively spliced isoforms that differ within the catalytic domain, but their accurate quantification is complicated by the highly homologous paralog ASAH2B. We developed an RT-qPCR-based method that combines a universal primer set for total ASAH2 with an isoform 1-specific primer set, enabling differential quantification of ASAH2 isoforms without detectable amplification of the homologous ASAH2B paralog. Absolute quantification was performed using standard curves generated from sequence-verified amplicons. Both assays demonstrated linear amplification over a range of 10²-10⁷ copies, with high correlation coefficients (r² = 0.999 for both assays). Melting curve analysis and agarose gel electrophoresis confirmed single, specific amplification products. Application of this assay to human CRC cell lines revealed that isoform 2, estimated by subtracting the isoform 1 copy number from the total ASAH2 copy number, was the predominant ASAH2 transcript across all tested cell lines. This assay provides a practical approach for transcript-level profiling of ASAH2 isoforms in colorectal cancer cell models and may support future studies of ASAH2 isoform regulation in tumor-related laboratory investigations.