Sybr green qpcr assay kit

Total RNA was extracted using the TRIzol^® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), which was then converted to cDNA using a Taqman^® miRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The expression of miR was determined using a Hairpin-it miRNAs qPCR Quantitation kit (Shanghai GenePharma Co, Ltd., Shanghai, China). U6 was used as the internal reference. The expression of mRNA was examined using a SYBR^® Green qPCR Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. GAPDH was used as the internal reference for mRNA. The thermocycling conditions were initial denaturation at 95°C for 10 min, then 40 cycles at 95°C for 15 sec and annealing/elongation at 60°C for 15 sec. The relative expression was analyzed by the 2^−ΔΔCq method (22 (link)). The primers for miR-23a and U6, which was used as the internal reference for microRNAs, were purchased from Fulgene (Guangzhou, China; cat nos. HmiRQP0345 and HmiRQP9001, respectively). The primer sequences were as follows: miR-23a forward, 5′-CCTACTGTCGTCCCAAGACCT-3′ and reverse, 5′-GGGGCTCGTGCAGAAGAAT-3′; U6 forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′. GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.

cDNA synthesis was performed with 1 µg total RNA (21 (link)) using the High Capacity Reverse Transcription kit (Applied Biosystems). The cDNA samples were amplified using the SYBR Green qPCR assay kit (Applied Biosystems) and the StepOnePlus real-time PCR system (Applied Biosystems). Primers used for TIGAR analysis are: forward, 5′-CTCCAGTGATCTCATGAG-3′; reverse, 5′-AGACACTGGCTGCTAATC-3′.