qRT-PCR

Isolation of total RNA was conducted using RNeasy Mini Kit (Qiagen). Reverse transcription was conducted via PrimeScript^TM RT reagent Kit (Takara, Shiga, Japan) as per the protocol specified by the manufacturer. Quantification of relative gene expression was conducted through qRT-PCR with SYBR Premix ExTaq kit (Takara, Shiga, Japan). One microgram of RNA was reverse-transcribed using an M-MLV Kit (Takara, Shiga, Japan) and random 9-mer primers (Takara, Shiga, Japan). Semiquantitative PCR was conducted using 50 ng of reverse-transcribed cDNA and 0.4 μM of each primer in a final reaction volume of 20 µL containing 1× PCR master mix (Takara, Shiga, Japan). GAPDH was used for expression normalization via the 2^−ΔΔCt method. The used primer sequences are itemized in Table 1.

Primer Sequences for Quantitative Real-Time PCR