Quantitative Analysis of Brown Adipocyte Markers

First-strand cDNA of HB2 brown adipocytes was obtained by incubating total RNA samples (2 μg) with reverse transcriptase (PrimeScrip II 1st strand cDNA Synthesis Kit, Takara Bio, Kusatsu, Japan) in a reaction mixture (20 μL). The RT product was amplified using a KAPA SYBR Fast qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) in triplicate in a 7500 Real Time PCR System (Applied Biosystems). PCR conditions were 50 °C for 2 min and 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. An 18s rRNA gene was used as an internal control, and the primer sequences were referenced from previous studies. Quantitative real-time PCR was performed for brown adipocyte marker genes such as 18S rRNA, Lgals3, Lgals3bp, UCP1, PGC-1α, Dio2, Elovl3, and C/EBPα using their specific primers. Each primer sequence was referenced in previous studies [28 (link),43 (link),62 (link),63 (link)]. The 18S rRNA was used as an internal control.
The mtDNA in fully differentiated HB2 transfectants was extracted using an mtDNA Extractor CT kit (Wako) according to the manufacturer’s instructions. The extracted mtDNA was amplified using the PCR protocol described above with primer sets that were reported to specifically amplify mouse mitochondrial DNA [44 (link)]. Each value was corrected for the corresponding nDNA (β2- macroglobulin DNA) value.