Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)
Table 2
Gene name, GenBank accession numbers, primer sequences, annealing temperature (), amplicon length, and PCR efficiency for the studied genes.
Genes
Accession number
Primers 5′-3′ (forward, reverse)
Amplicon size (bp)
PCR efficiency (%)4
A2M
1
CR452243
CACCCAGGACACAGTGGTAGC CCCTGAAGACTGGATGGTCAC
60°C
103
97.20
ACTB
1
AY141970
GCGTGGCTACAGCTTCACC TTGATGTCACGGACGATTTC
60°C
56
90.70
B2M
NM_173893
CTGCTATGTGTATGGGTTCC GGAGTGAACTCAGCGTG
60°C
101
131.37
EEF1A1
BC105315
CATCCCAGGCTGACTGTGC TGTAAGCCAAAAGGGCATGC
60°C
101
115.50
GAPDH
1
BC102589
TGGAAAGGCCATCACCATCT CCCACTTGATGTTGGCAG
60°C
60
96.64
GTP
1
AK074976
CTTGGAATCCGAGGAGCCA CCTGGGATCACCAGAGCTGT
60°C
101
102.91
HMBS
2
BC112573.1
CTTTGGAGAGGAATGAAGTGG AATGGTGAAGCCAGGAGGAA
60°C
80
101.4
HPRTI
3
BC103248
GAGAAGTCCGAGTTGAGTTTGGAA GGCTCGTAGTGCAAATGAAGAGT
64°C
190
99.70
RPL22
1
BC114880
AAGATGGCGCCGAAGAAAG TTTCCCGAATCAAAAATTCCA
60°C
101
102.73
RPL4
NM_001014894
TTGGAAACATGTGTCGTGGG GCAGATGGCGTATCGCTTCT
60°C
101
93.72
RPS15A
BC108231
GAATGGTGCGCATGAATGTC GACTTTGGAGCACGGCCTAA
60°C
101
119.08
RS18
DQ222453.1
TTGCCTTTGCCATCACTG CTTGTATTGGCGTGGATT
60°C
158
129.97
RPS23
1
BC102049
CCCAATGATGGTTGCTTGAA CGGACTCCAGGAATGTCACC
60°C
101
115.5
RPS9
1
DT860044
CCTCGACCAAGAGCTGAAG CCTCCAGACCTCACGTTTGTTC
60°C
54
96.45
UBC
2
BE668033
TCCCTACCTGCATCATGTGC GGAATTTGGGCCAGTGCTC
59°C
71
104.71
UXT
1
CR452243
TGTGGCCCTTGGATATGGTT GGTTGTCGCTGAGCTCTGTG
60°C
101
99.74
1Bionaz and Loor [19],2Pérez et al. [32], and 3Hernandez et al. [33].
4qPCR efficiencies for each primer pair were calculated from six-point standard curves using fivefold dilution series of pooled cDNA from control and heat-stressed samples.
