Gpr120, Fshb, Lhb, and Gnrh-r mRNA expression levels were determined by real-time PCR using the SYBR® Premix Ex
Taq™ II master mix (Takara Bio, Shiga, Japan) containing SYBR® Green I, and run on the 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Denaturation was
performed at 95°C (30 sec), amplification was conducted for 40 cycles with denaturation at 95°C (5 sec), and annealing and amplification were conducted at 60°C (34 sec). Data were
analyzed using the standard curve method [22 (link)]. The forward and reverse primer set (Nippon EGT, Toyama, Japan) sequences used for each mouse
gene are shown inTable 1 List of primer sequences for real-time PCR
for the presence of a single peak in the dissociation curve.
Taq™ II master mix (Takara Bio, Shiga, Japan) containing SYBR® Green I, and run on the 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Denaturation was
performed at 95°C (30 sec), amplification was conducted for 40 cycles with denaturation at 95°C (5 sec), and annealing and amplification were conducted at 60°C (34 sec). Data were
analyzed using the standard curve method [22 (link)]. The forward and reverse primer set (Nippon EGT, Toyama, Japan) sequences used for each mouse
gene are shown in
| Gene | Forward primer | Reverse primer |
|---|---|---|
| Gpr120 | 5’-TCGCTGTTCAGGAACGAATG-3’ | 5’-CACCAGAGGCTAGTTAGCTG-3’ |
| Lhβ | 5’-CTAGCATGGTCCGAGTACTG-3’ | 5’-CCCATAGTCTCCTTTCCTGT-3’ |
| Fshβ | 5’-CTGCTACACTAGGGATCTGG-3’ | 5’-TGACATTCAGTGGCTACTGG-3’ |
| Gnrh-r | 5’-CAGGATGATCTACCTAGCAG-3’ | 5’-GCAGATTAGCATGATGAGGA-3’ |
| L19 | 5’-CCAAGAAGATTGACCGCCATA-3’ | 5’-CAGCTTGTGGATGTGCTCCAT-3’ |
for the presence of a single peak in the dissociation curve.