RNA was isolated with Trizol (Invitrogen, Cat# 15596026) and chloroform, as previously described [47 ]. In brief, the cells were lysed in Trizol, after washing with ice-cold PBS. RNA was isolated with chloroform, precipitated with isopropanol, washed twice with ethanol, dried, and resuspended in water. The quality and concentration of the RNA were assessed using a Nanodrop2000 (Thermo Fisher Scientific).
For reverse transcription, we used 0.5 μg RNA and the Omniscript RT Kit (Qiagen, Cat# 205111) with a “random” primer (Roche, Cat# 11034731001), following the manufacturer’s protocol. For real-time PCR, we diluted 20 µl cDNA preparation with 480 µl water and used 9 µl of the dilution together with 11 µl of the Taqman Gene Expression Master Mix (Applied Biosystems, Cat# 4440040), and the respective Taqman probes for SMPD1, SMPD2, SMPD3, CERS5, CERS6, and GAPDH (all from Thermo Fisher Scientific: SMPD1 Cat# 4331182, Assay ID Hs04183448_m1; SMPD2, Cat# 4331182, Assay ID Hs00162006_m1; SMPD3, Cat# 4331182, Assay ID Hs00920354_m1; CerS5, Cat# 4331182, Assay ID Hs00332291_m1; CerS6, Cat# 4331182, Assay ID Hs00826756_m1; GAPDH, Cat# 4331182, Assay ID Hs02786624_g1) in a 7500 HT real-time PCR instrument (Applied Biosystems). We used GAPDH as an internal control and relative expression was calculated as 2−ΔΔCT.
For reverse transcription, we used 0.5 μg RNA and the Omniscript RT Kit (Qiagen, Cat# 205111) with a “random” primer (Roche, Cat# 11034731001), following the manufacturer’s protocol. For real-time PCR, we diluted 20 µl cDNA preparation with 480 µl water and used 9 µl of the dilution together with 11 µl of the Taqman Gene Expression Master Mix (Applied Biosystems, Cat# 4440040), and the respective Taqman probes for SMPD1, SMPD2, SMPD3, CERS5, CERS6, and GAPDH (all from Thermo Fisher Scientific: SMPD1 Cat# 4331182, Assay ID Hs04183448_m1; SMPD2, Cat# 4331182, Assay ID Hs00162006_m1; SMPD3, Cat# 4331182, Assay ID Hs00920354_m1; CerS5, Cat# 4331182, Assay ID Hs00332291_m1; CerS6, Cat# 4331182, Assay ID Hs00826756_m1; GAPDH, Cat# 4331182, Assay ID Hs02786624_g1) in a 7500 HT real-time PCR instrument (Applied Biosystems). We used GAPDH as an internal control and relative expression was calculated as 2−ΔΔCT.