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. 2014 May 7;2014:382653. doi: 10.1155/2014/382653

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.