Thunderbird qpcr mix

The cells were cultured in 100-mm diameter dishes or six-well plates, and total RNA was extracted from cells using ISOGEN (NIPPON GENE CO., Tokyo, Japan) or Nucleospin RNA^® (Macherey-Nagel GmbH & Co. KG, Düren, Germany). cDNA was synthesized by ReverTra Ace^® (TOYOBO). The RT-qPCR was done using THUNDERBIRD^® qPCR Mix (TOYOBO) and Step One Plus™ (Thermo Fisher Scientific). Gene expression was quantified and then normalized against the RPL30 housekeeping gene expression10 (link). The primer sequences were as follows: endogenous DKK3, CAG GCT TCA CAG TCT GGT GCT TG (forward) and ACA TTG TTT CCA TCT CCT CCC CTC (reverse)11 (link); DKK3-HA-Tag, AGG AAC TGA TGG AGG ACA CG (forward) and CTT CTG CCT TCT TCG TCT CC (reverse); RPL30, ACA GCA TGC GGA AAA TAC TAC (forward) and AAA GGA AAA TTT TGC AGG TTT (reverse); cyclin D1, CTT CCT CTC CAA AAT GCC AG (forward) and AGC GTG TGA GGC GGT AGT AG (reverse); and c-myc, TCC TCG GAT TCT CTG CTC TC (forward) and GTT GTG CTG ATG TGT GGA GA (reverse). The PCR conditions were 95°C for 1 min followed by 40 cycles of 95°C for 15 s and 60°C for 45 s. Absolute quantification was used for analysis as previously described12 (link).

Total RNA was isolated from the hypothalamus of each of the control animals (n=8, 12 weeks old), HFD animals (n=8) at ZT6, P0 embryos in the mock group (n=4) or P0 embryos in the PLAGL1 group (n=6) using Sepasol-RNA I Super G reagent (Nacalai Tesque, Inc., Kyoto, Japan). Single-stranded cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan), according to manufacturer's protocol. The expression levels of each mRNA were determined by qPCR with the Rotor-Gene Q system (Qiagen GmbH, Hilden, Germany) using the THUNDERBIRD qPCR mix (Toyobo Life Science) and gene-specific primers (Table SI). PCR products were amplified using the following thermocycling conditions: One cycle at 95°C for 1 min, followed by 40 cycles at 95°C for 10 sec and 60°C for 60 sec. The housekeeping gene with minimum diurnal variation in the hypothalamus was identified in a previous study by checking gene expression every 4 h over a 24-h period (23 (link)). Subsequently, the relative level of target gene expression was evaluated using the 2^-ΔΔCq method (24 (link)) with hypoxanthine phosphoribosyltransferase 1 as an internal control.

Total RNA was extracted from resected specimens and cultured cells as described above, and reverse transcription was undertaken using the PrimeScript RT Reagent kit (Takara Bio, Otsu, Japan). The cDNA was amplified by qRT‐PCR on a Thermal Cycler Dice Real Time System II (Takara Bio) using the Thunderbird qPCR Mix (Toyobo Life Science, Osaka, Japan). Sequences of primers used for amplification are shown in Table S1.