RT-qPCR

RCAs freshly isolated from diabetic or non-diabetic rats were used for RT-qPCR analysis. RT-qPCR was used to detect transcript levels as previously described (32-34). SV Total RNA Isolation System (Promega Corporation) was used to isolate total RNA from the RCAs in accordance to the manufacturer's protocol. A microplate reader (BioTek Instruments, Inc.) was used to measure the concentration and purity of the RNA samples. A total of 1 µg RNA was added into the reaction system (PrimeScript™ RT reagent kit with gDNA Eraser; Takara Bio, Inc.) comprising of 2 µl 5X gDNA Eraser Buffer, 1 µl gDNA Eraser and appropriate volumes DEPC-treated water to remove genomic DNA. The mixture was then incubated for 2 min at 42˚C. Total RNA was converted into cDNA in a 20 µl PCR reaction system with 10 µl reaction system from the previous step, 1 µl PrimeScript RT Enzyme Mix I, 1 µl RT Primer Mix, 4 µl 5X Primer Script Buffer 2, 4 µl RNase Free dH₂O (PrimeScript^™ RT reagent kit with gDNA Eraser; Takara Bio, Inc.). C1000 Touch^™ Thermal cyclers (Bio-Rad Laboratories, Inc.) were used to perform RT with the following heat cycle: 37˚C for 15 min and 85˚C for 5 sec. A total of 2 µl cDNA was amplified in a 23 µl reaction system containing 12.5 µl SYBR^® Premix Ex Taq^™ (Takara Bio, Inc.), 8.5 µl DEPC-treated water, 1 µl forward primer (10 µM) and 1 µl reverse primer (10 µM). The following thermocycling conditions were used for the PCR: Initial denaturation at 95˚C for 30 sec; followed by 40 cycles of 95˚C for 5 sec and 60˚C for 30 sec. The melting curve included an increase from 65˚C to 95˚C for 5 sec. The following primer pairs were used for the qPCR: Kv1.2 forward, 5'-TGTCTATCACCCAGGAACATGGAG-3' and reverse, 5'-GAGCTTGGGTCTGAAGCCTTTG-3'; Kv1.5 forward, 5'-CCTCCGACGTCTGGACTCAATAA-3' and reverse, 5'-CCTCATCCTCAGCAGATAGCCTTC-3' and GAPDH forward, 5'-GGCACAGTCAAGGCTGAGAATG-3' and reverse, 5'-ATGGTGGTGAAGACGCCAGTA-3' (Takara Bio, Inc.). Relative gene expression was calculated using the 2^-ΔΔCq method (35).