Gene Expression Analysis of Adipogenesis

The total RNA (1 μg) extracted from the cells on day 6 of the maturation phase using acid guanidium thiocyanate/phenol/chloroform was reverse transcribed (RT) using M-MLV reverse transcriptase (Ribonuclease H Minus Point Mutant). The single-stranded cDNA was synthesized using oligo-(dT)₁₅ and a random 9-mer (Promega Corp., Madison, WI, USA) as primers in the RT reaction. The amount of transcript was determined via qRT-PCR using TB GreenTM Premix Ex TaqTM II (Tli RNaseH Plus) kits (Takara Bio Co., Inc., Kusatsu, Japan) and a Thermal Cycler Dice^TM Real Time System (Takara Bio Co., Inc., Kusatsu, Japan) according to the threshold cycle (CT) and ^ΔΔCT methods described by the manufacturer. Table 1 shows the oligonucleotides used herein. The cycling program comprised 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 30 s, followed by 95 °C for 15 s and 60 °C for 30 s. Amounts of target gene transcripts were normalized to those of β-Actin. The accession numbers of the target genes are as follows: PPARγ, NM_011146; adiponectin, NM_009605.5; LPL, NM_008509.2; DP1, NM_008962.4; DP2, XM_006526696.5; β-Actin, NM_007393.