Mini micro rna isolation 1 kit

Total RNA was extracted from MGCs or CGCs using a Mini/Micro RNA Isolation I kit (Zymo Research, CA, USA) according to the manufacturer's instructions. RNA purity and concentration were assessed using a NanoDrop spectrophotometer (NanoDrop 2000C, Thermo Scienti c Waltham, MA, USA). Total RNA (25ng) from each sample was used for cDNA synthesis by a high capacity reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's instructions in a 10μl total volume reaction. mRNA levels were analyzed by real-time PCR using the StepOnePlus real-time PCR system (Applied Biosystems). The real-time PCR mix contained 1μl of cDNA, fast SYBR Green Master Mix (Applied Biosystems), and speci c primers for ABCC4 or other gene of interest and β-actin (housekeeping gene) in a total volume of 10μl. Cycling parameters were: 1 cycle at 95°C for 20 seconds, and 40 cycles each at 95°C for 3 seconds and at 60°C for 30 seconds. A melting curve analysis was performed at the end of each run to ensure measurements were based on the ampli cation of the target gene. All samples were run in duplicate. Analysis of the qPCR results was carried out with StepOne software. Relative gene expression was calculated using the delta-delta Ct method. Details of the primers used are shown in Table S1.