Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Germany, 74,034) according to the manufacturer’s protocol. Then, RNA concentration was determined by NanoDrop 2000 (Thermo Fisher) and cDNA was obtained from total RNA (50 ng) of each sample by reverse transcription using oligo (dT)15 and reverse transcriptase (Promega, USA, A3500), according to the manufacturer’s instructions, at 42 °C for 15 min, followed by 95 °C for 5 min in 20 μl total volume.
Quantitative real-time PCR was performed using SYBR Green Master Mix (ABI, Germany, DBI-2044) in an Applied Biosystems 7500 Real-Time PCR System. An aliquot (10%) of cDNA was subjected to 40 amplification cycles of PCR with the primers listed in Supplementary Table 1. Cycling parameters were one cycle for 2 min at 50 °C, one cycle for 10 min at 95 °C, 40 cycles at 95 °C for 15 s, and 60 °C for 20 s. For each experiment, three replicates were included in each qPCR reaction. We performed melting curve analysis at the end of each run to ensure a single amplicon.
The relative levels of endogenous β-actin mRNA (ACTB) mRNA were used as an internal control [40 (link)], and data were analyzed using the 2-(△△Ct) method. Only samples whose cycle threshold (Ct) values of ACTB were between 21 and 22 were included in the analysis.
Quantitative real-time PCR was performed using SYBR Green Master Mix (ABI, Germany, DBI-2044) in an Applied Biosystems 7500 Real-Time PCR System. An aliquot (10%) of cDNA was subjected to 40 amplification cycles of PCR with the primers listed in Supplementary Table 1. Cycling parameters were one cycle for 2 min at 50 °C, one cycle for 10 min at 95 °C, 40 cycles at 95 °C for 15 s, and 60 °C for 20 s. For each experiment, three replicates were included in each qPCR reaction. We performed melting curve analysis at the end of each run to ensure a single amplicon.
The relative levels of endogenous β-actin mRNA (ACTB) mRNA were used as an internal control [40 (link)], and data were analyzed using the 2-(△△Ct) method. Only samples whose cycle threshold (Ct) values of ACTB were between 21 and 22 were included in the analysis.
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