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Gene expression analysis

HO Hirofumi Okada
CN Chiaki Nakanishi
SY Shohei Yoshida
MS Masaya Shimojima
JY Junichiro Yokawa
MM Masayuki Mori
HT Hayato Tada
TY Tsuyoshi Yoshimuta
KH Kenshi Hayashi
TY Tomoyoshi Yamano
RH Rikinari Hanayama
MY Masakazu Yamagishi
MK Masa-aki Kawashiri
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Total RNA was isolated from cells using an RNAeasy kit (Qiagen). For real-time PCR analysis, cDNA was synthesized using SuperScript III reverse transcriptase and oligo (dT) primers (Invitrogen). Primers used for amplification are listed in Supplemental Table 1. Real-time PCR was conducted using the Power SYBR® Green PCR Master Mix (Applied Biosystems). Real-time PCR for LDLR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed using an Mx3000P QPCR system (Agilent Technologies, CA, USA), and the cycling conditions were 10 min at 95 °C, followed by 45 cycles of 30 s at 95 °C and 1 min at 60 °C. Cycle threshold was calculated under default settings using real-time sequence detection software (Agilent Technologies). The expression levels of LDLR were normalized to those of GAPDH.

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