Sybr green primescript rt kit

The miR-21-5p, miR-34c-5p, miR-449a-5p and U6 primers were designed by Boheng Biomart (Beijing, China). Total RNA from AEC-II cells was isolated using the RNAiso Kit (Takara Biotechnology Co., Ltd., Dalian, China). RNA concentration and purity was checked using a Varioskan Flash spectrophotometer (Thermo Fisher Scientific, Inc.). A total of 500 ng RNA was reverse transcribed to cDNA using the PrimeScipt RT Reagent Kit (Takara Biotechnology Co., Ltd.) in a final volume of 10 µl, according to the manufacturer's protocol. RT-PCR was performed with a CFX Connect Real-Time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a SYBR green PrimeScript RT kit (Takara Biotechnology Co., Ltd.). and the following thermocycling conditions: Initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 sec and combined annealing/extension at 65.7°C for 30 sec. The primer sequences were as follows: miR-34c-5p forward, 5′-CCAGGCAGTGTAGTTAGCT-3′ and reverse, 5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACGCAATC-3′; miR-449a-5p forward, 5′-AACGTGGCAGTGTATTGTTA-3′ and reverse, 5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACCAGC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACACG-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. U6 was used as an internal control. The relative expression levels were calculated using the 2^−ΔΔCq method (21 (link)).