Real-Time PCR Analysis of Sphingomyelinase Genes

The total RNA of U251MG and HASTR/ci35 cells was extracted using a QIAshredder and an RNeasy^® Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. The pairs of primers and the TaqMan probes for the target mRNAs were designed based on the human mRNA sequence using TaqMan^® Gene Expression Assays (Assay ID: sphingomyelinase 1 (SMPD1), Hs03679346_g1; SMPD2, Hs00906924_g1; SMPD3, Hs00920354_m1; SMPD4, Hs04187047_g1; SMPD5, Hs04994298_g1; SMPDL3A, Hs01041066_m1; SMPDL3B, Hs01038741_m1; survivin (also known as BIRC5), Hs00977612_mH; GAPDH, Hs99999905_mL, Applied Biosystems, Foster City, CA, USA). For one-step real-time PCR, 20 ng of total RNA was added to the master mix of the TaqMan^® RNA-to-CT^TM 1-Step Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The RNA was then analyzed using the LightCycler^® 96 system (Roche Diagnostics, Mannheim, Germany). The relative mRNA expression levels of the target genes in cells were calculated using the comparative threshold cycle (Ct) method, according to previous studies [11 (link),12 (link)]. The mRNA expression level relative to GAPDH for each target PCR was calculated using the following equation: relative mRNA expression = 2^{−(Ct target − Ct GAPDH)} × 100% [11 (link),12 (link)].