Quantification of Cytokine Expression

The mammary gland tissues were homogenized in PBS, frozen and thawed three times; the supernatants were obtained after centrifugation at 9000× g for 20 min. The total RNA was then extracted from the supernatants of the mammary gland tissues by the TRIzol reagent, and the cDNA was generated by using a reverse transcription kit (Invitrogen, CA, USA) according to the manufacturer’s instructions. The specific primers of five cytokines were listed in Table 1 [35 (link),40 (link),41 (link),42 (link)]. Quantitative real-time PCR was performed on an ABI 7300 Real-Time PCR Detection System (Applied Biosystems, Foster, CA, USA) in a 20-μL reaction volume. Each sample was assessed in triplicate. The reaction volume and the reaction condition can be referenced from our lab’s previous studies [29 (link)]. The results (fold changes) were normalized by GAPDH using the 2^−ΔΔCt comparative method [41 (link)].