RT-qPCR analysis

RT-qPCR was performed to detect the expression of PDE4A, B, C and D genes. Total RNA was isolated from cells using TRIzol reagent and a spectrophotometer was used to determine the quality and quantity of the RNA. TRIzol reagent was used according to the manufacturer's instructions. In brief, a total of 250 µl TRIzol reagent was added into the well (5×10⁵ cells/well) to lyse the cells by pipetting them up and down several times after removing the growth media from the well. Next, 50 µl chloroform was added after incubating the homogenized sample for 5 min in the tube at room temperature. Following vigorous shaking for 15 sec and incubation at room temperature for 3 min, the sample was centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase of the sample was removed into a new tube, then incubated with 125 µl isopropanol at room temperature for 10 min. The total RNA was precipitated by centrifugation at 12,000xg for 15 min at 4°C. The RNA pellet was washed with 250 µl 75% ethanol, then centrifuged at 7,500xg for 5 min at 4°C. After discarding the wash, the RNA pellet was air dried for 8 min. Finally, the RNA pellet was suspended in RNase-free water (20 µl). The isolated RNA was stored at −70°C before RT-qPCR. The quality and quantity of RNA were determined using a spectrophotometer by measuring the OD levels at 260 nm and 280 nm, respectively. The ratios of OD 260/OD 280 for the samples were between 1.8–2.0, which indicated the purity of the samples was quite good. Thus we did not treat the isolated RNA before RT-qPCR.

cDNA was synthesized from the RNA using reverse transcription. The reverse transcription (RT) reaction was performed in a 20 µl total reaction volume containing 4 µl of 5X RT buffer, 4 µl of 2.5 mM dNTPs, 1 µl of Multiscribe reverse transcriptase (50 U/µl) (Promega, Madison, WI, USA), 1 µl of RNase inhibitor, 5 µl of RNase-free water and 3 µg of isolated total RNA in a 5 µl volume. The RT reaction was performed at 25°C for 10 min, 37°C for 120 min and 95°C for 5 min. qPCR analysis was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed in a final volume of 20 µl, containing 10 µM each primer, 1 µl SYBR green (20X) and 1 µl template. The program profile was as follows: 95°C for 2 min, 40 cycles of denaturation for 10 sec at 95°C, annealing for 30 sec at 60°C and extension for 30 sec at 70°C. The PCR primers used in this study are summarized in Table II.

PCR primers used in the present study.

PCR, polymerase chain reaction; PDE, phosphodiesterase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

The ΔΔCq method was used to normalize the qPCR data, and the level of GADPH mRNA was utilized as a reference. The relative expression level of target mRNA was calculated according to the difference between the reference and target Cq values for each sample. Next, the level of target mRNA in groups with different treatment was normalized by setting the level in the control group as 1.