Functional Interactions between 17β-Estradiol and Progesterone Regulate Autophagy during Acini Formation by Bovine Mammary Epithelial Cells in 3D Cultures

Table 1

Primer sequences of examined ATGs and parameters of real-time PCR assay.

Target gene	Nucleotide sequence	Real-time PCR
ATG5	FRD: 5′-TTT GAA TAT GAA GGC ACA CC-3′	SYBR Select Master Mix; (catalogue number 4472908; Applied Biosystems); (i) 50°C for 2 min; (ii) 95°C for 2 min; (iii) 40 cycles of; (a) 95°C for 15 sec; (b) 58°C for 15 sec; (c) 72°C for 1 min
	REV: 5′-TGT AAA CCC ATC CAG AGT TG-3′
ATG3	FRD: 5′-GGT TGT TCG GCT ATG ATG AG-3′
	REV: 5′-GGG AGA TGA GGG TGA TTT TC-3′
BECN1	FRD: 5′-AGT TGA GAA AGG CGA GAC AC-3′
	REV: 5′-GAT GGA ATA GGA ACC ACC AC-3′
MAP1 LC3 B	FRD: 5′-TTA TCC GAG AGC AGC ATC C-3′
	REV: 5′-AGG CTT GAT TAG CAT TGA GC-3′
GAPDH	FRD: 5′-CTT CAA CAG CGA CAC TCA-3′
	REV: 5′-CCA GGG ACC TTA CTC CTT-3′

Primers were designed using Primer 3 software, on the basis of the bovine sequences from NCBI database and verified using Oligo Calc: Oligonucleotide Properties Calculator (free software available online, provided by Northwestern University) to exclude sequences showing self-complementarity, and BLAST (NCBI, U.S. National Library of Medicine) to exclude possible complementarity to other mRNA templates.