Abi prism 7500 rt qpcr system

Total RNA was separated with TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). To verify expression results, 2 μg of total RNA was used for cDNA synthesis with a Takara PrimeScript RT Reagent kit (Takara, Shiga, Japan). A housekeeping glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. The cDNA was used for RT-qPCR with SYBR Green Master Mix (Takara, Shiga, Japan) on an ABI PRISM 7500 RT-qPCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Primers were list in Supplementary Table S1. The relative expression of these RNAs was analyzed with the 2^−ΔΔCt method.

Total RNA content was extracted using the TRIzol reagent (15596026, Invitrogen), and then reverse-transcribed into cDNA utilizing PrimeScript Reverse Transcription Reagent kits (RR047A, Takara, Japan). For miRNA detection, the extracted RNA was reverse-transcribed into cDNA with the help of a miRNA-specific stem-loop RT primer. RT-qPCR was subsequently carried out using Fast SYBR Green PCR kits (Applied Biosystems Inc., Carlsbad, CA, USA) on an ABI PRISM 7500 RT-qPCR system (Applied Biosystems). At least 3 repeats were set for each well. U6 was adopted as the loading control for miR-1-3p, and GAPDH for MALAT1 and VASP. The primers are illustrated in Supplementary Table 1. The 2^−ΔΔCt method was utilized for quantifying relative expression.