Lightcycler 480 rt pcr detection system

Total RNAs were extracted using TRIzol reagent (Invitrogen, USA). One µg of total RNA from the samples was reverse transcribed using a Reverse Transcription Kit (BioRad, Hercules, CA, USA). RT-PCR was performed using SYBR Green (BioRad) in the LightCycler 480 RT-PCR Detection System (Roche). Primers were synthesized by Sangon Biotech Company (Shanghai, China): SLCO4A1-AS1 forward 5'-CACTTTCCAGCCTCTCACCA-3', and reverse 5'-GGCCACCTCCTCAAACAAGA-3'; β-actin forward 5'-TCACCAACTGGGACGACATG-3', and reverse 5'-GTCACCGGAGTCCATCACGAT-3'. SLCO 4A1-AS1 expression was normalized to the respective β-actin expression level. Relative expression was calculated using the equation: ΔCt = Ct (target gene) - Ct (β-actin), fold expression = 2^{-(ΔCt(tumor) -} ΔCt(normal)) by Cq value.

Total RNA was extracted using TRIzol reagent according to the manufacturer’s protocol (Invitrogen, USA). cDNA was synthesized using a reverse transcription kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed using SYBR Green (Bio-rad, Hercules, CA, USA) with a LightCycler 480 RT-PCR detection system (Roche). β-actin was used as an internal control for normalization. The expression of DNAJC3-AS1 was normalized to the corresponding β-actin expression level. The following equation was used to calculate the relative expression: ΔCt = Ct (target gene) − Ct (β-actin), fold expression = 2 – [ΔCt (tumor) − ΔCt (normal)] divided by the Cq value. The primer sequences used in the experiment were as follows: DNAJC3-AS1: 5′-AGCGATTGTGGAAGACCCTG-3′, 5′-ATTTCCCCTGGTAAGCGCAA-3′ and β-actin: 5′-TCACCAACTGGGACGACATG-3′, 5′-GTCACCGGAGTCCATCACGAT-3′.