Mammary Tissue RNA Extraction and qPCR

Total RNA was extracted from the mammary tissues using the RNA Pure Kit according to the manufacturer's procedures (Aidlab Biotechnologies Co., Ltd, Beijing, China). Total RNA of 1000 ng was reverse transcribed to cDNA using the PrimeScript RT reagent (Takara, Tokyo, Japan). Primers for qPCR were designed using the National Center for Biotechnology Information Primer-BLAST site and listed in Supplemental Table 1. qPCR was performed in a 7500c real-time PCR detection system (Applied Biosystems, Carlsbad, California, USA) using SYBR premix EX Taq (Takara). SDHA, HPRT1, and ARBP were selected as reference genes from six housekeeping genes (SDHA, HPRT1, ARBP, GAPDH, β-actin, and B2M) using geNorm [13 (link), 14 (link)]. The 2^−ΔΔCt method was used to calculate the relative mRNA expression of genes.