Taqman fast plate

Vascular smooth muscle cells (CADASIL and control) were grown overnight in a six-well plate chamber with confluence of 100,000 cells. The following day, the cells were lysed with RIPA buffer (Thermo Fisher Scientific, United States) and the quality of RNA was determined with RIN (RNA Integrity Number) of 10. cDNA was prepared using Taqman gene expression master mix (Applied Biosystem) and SuperScript VILO cDNA Synthesis kit (Thermo Fisher Scientific, United States) according to the manufacturer’s protocol. Quantitative (q)RT-PCR was performed using costume format TaqMan fast plate (Applied Biosystems; No. 4427562, Rev C)¹. The gene designations for control and GLUTs were: HPRT1-Hs99999909_m1 (endogenous control), GAPDH-Hs99999905_m1 (endogenous control), 18S-Hs99999901_s1, SLC2A2-Hs01096908_m1; SLC2A3-Hs00359840_m1, and SLC2A4-Hs00168966_m1. All probes were used in duplicates with 30 ng of cDNA. Quantitative RT-PCR was performed on a 7500 Fast Real-Time PCR System (Life Technologies, United States). The expressions of genes were normalized to internal control HPRT gene and analysis was used comparing the normalized value of control cell line to the normalized values of CADASIL cell line. All the quantitative data were from three independent biological replicates for each experiment and the control value was normalized to 1.

VSMCs were grown overnight in a 6-well plate chamber with confluence of 100,000 cells. The following day, cells were lysed with RIPA buffer (ThermoFisher Scientific, U.S.A.) and the quality of RNA was determined with RIN (RNA Integrity Number) of 10. cDNA was prepared using Taqman gene expression master mix (Applied Biosystem) and SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific, U.S.A.) according to the manufacturer’s protocol. Quantitative (q)RT-PCR was performed using costume format TaqMan fast plate (Applied Biosystems; No. 4,427,562, Rev C; https://www.thermofisher.com/order/taqman-files). The gene designations for control were: HPRT1-Hs99999909_m1 (endogenous control) and GAPDH-Hs99999905_m1 (endogenous control). All probes were used in duplicates with 30 ng of cDNA. Quantitative RT-PCR was performed on a 7500 Fast Real-Time PCR System (Life Technologies, U.S.A.). The expressions of genes were normalized to internal control HPRT gene and analysis was used comparing the normalized value of control cell line to the normalized values of CADASIL cell line. All the quantitative data were from three independent biological replicates for each experiment and the control value was normalized to 1.