Quantitative Analysis of Gene Expression

Total RNA was isolated using a FastGene RNA Premium Kit (NIPPON Genetics Co., Ltd., Bunkyo, Tokyo, Japan) according to the manufacturer’s instructions. cDNA was synthetized using FastGene Scriptase II (NIPPON Genetics Co., Ltd.). Real-time PCR was performed using the Thermal Cycler Dice Real Time System (Takara Bio, Inc., Otsu, Shiga, Japan) with a KAPA SYBR Fast qPCR Kit (Roche Diagnostics K.K., Minato, Tokyo, Japan). Primer sequences for quantification of Prkar1b cDNA were 5′-CTTCAGTCCTCCAGCGACGAT-3′ and 5′-GGTTCAGCAGCAGGGCAAT-3′, and those for Sun1 cDNA were 5′-GAGTCAGGCAGGGATTTCAG-3′ and 5′-AGAGCACCGAGTGCTTAGGA-3′. The number of target molecules in a test sample was analyzed by monitoring the amplification curves for both test and reference samples, all of which contained 10¹ to 10⁶ molecules of the gene of interest. The number of target molecules was normalized to that of peptidylprolyl isomerase A (Ppia) as an internal control^{35 (link)}.