Ibright fl1000 image system

Then, total RNA was extracted from the cells by using the Trizol reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. The concentration and quality of RNA were determined using Qubit 3.0 (Thermo fisher). The extracted RNA was used as a template for subsequent cDNA synthesis with oligo dT primers using the Prime script RT reagent Kit (Thermo Fisher). Initial mRNA levels were quantified using a Premix Taq™ cDNA kit (Takara Bio Inc) in accordance with the manufacturer's instruction. DNA was first denatured at 98℃ for 10s and then annealed at 68°C for 30 seconds. This was repeated for 30 cycles before a final extension at 72℃ for 10 minutes. Then, the PCR products were run on a 2% agarose gel; gene expression was assessed with the use of iBright FL1000 image system (Thermo fisher Scientific, USA). The gels were quantified by densitometry using iBright analysis software (Thermo fisher Scientific) and normalized to GAPDH. All values are reported as means ± standard deviation (SD). The following primers were used for amplification: AQP3, forward 5′‐CAT CTA CAC CCT GGC ACA GA‐3′ and reverse 5′‐GGC TGT GCC TAT GAA CTG GT‐3′；GAPDH forward 5′‐ GGT GAA GGT CGG AGT CAA CG‐3′ and reverse 5′‐ CAA AGT TGT CAT GGA TGH ACC‐3′.