Table 1.
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.