Transcriptional Profiling of Adipocyte Differentiation

Total RNA (1 μg) extracted from the cells after 6, 24, and 48 h of the differentiation phase, and on day 6 of the maturation phase using acid guanidium thiocyanate/phenol/chloroform was reverse transcribed (RT) using M-MLV reverse transcriptase (Point mutation without Ribonuclease H activity). Single-stranded cDNA was synthesized using oligo-(dT)₁₅ and a random 9-mer (Promega) as primers in the RT reaction. Transcript levels were determined by RT-qPCR 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.) 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. Levels of target gene transcripts were determined and normalized to those of β-Actin. The accession numbers of the target genes are as follows: C/Ebpβ, NM_009883; C/Ebpδ, NM_007679; Pparγ, NM_011146; C/Ebpα, NM_001287523; Lpl, NM_008509; Glut4, AB008453; Leptin, NM_008493; β-Actin, NM_007393.