Preliminary experiments using rats aged 10, 24, 48, and 96 weeks indicated that leptin mRNA expression in 48- and 96-week-old rats was significantly higher than that in 10-week-old rats (Additional file
1: Figure S1). However, a previous study reported that SD rats developed a tumor early or late in life, over the age range of 494 to 798 days (approximately 71 to 114 weeks) at the time of first tumor observation [
16]. Therefore, to evaluate changes in cytokine expression due to aging, SD rats were divided into two age groups: the young group (10 weeks) and aged group (48 weeks) (
n = 10 each). Animals were anaesthetized first with isoflurane following by an intramuscular injection of a mixture comprising medetomidine, midazolam, and butorphanol tartrate into the upper limb. Blood was removed to prevent contamination with blood components before sacrificing the rats by cervical dislocation. Bilateral quadricep muscles were removed without the fascia and washed with phosphate buffered saline solution (PBS). TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the quadricep muscles, based on the manufacturer’s protocol. The RNA formed the template for first-strand cDNA synthesis, which was performed with SuperScript III RT (Invitrogen) in a reaction (final volume, 25 μL) comprising 2 μL cDNA, a specific primer set (0.2 μM final concentration), and 12.5 μL SYBR Premix Ex Taq (Takara, Shiga, Japan). Primers for
Il1b, Il6, Tnfa, MuRF1, Atrogin1, and
leptin were designed using Primer Blast and made by Hokkaido System Science Co., Ltd. (Sapporo, Japan). The primer sequences are listed in Table
1. The primer-amplified products were confirmed for specificity using melt curve analysis. Q-RT-PCR was conducted on a CFX-96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The Q-RT-PCR protocol was as follows: initial denaturation at 95 °C for 1 min, and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. mRNA expression levels of inflammatory cytokine (
Il1b, Il6, Tnfa), muscle breakdown-related marker (
MuRF1,
Atrogin1) and
leptin in the quadricep muscles were determined by normalizing to that of glyceraldehyde-3-phosphate dehydrogenase (
Gapdh) using the delta-delta Ct method. Relative expression was calculated using the mean of all control samples (samples from quadriceps muscles from the young group or vehicle-treated quadriceps muscle-derived cells in vitro).
Table 1
Sequences of the primers used in this study
Il6
| F | CCAGTTGCCTTCTTGGGACT | 224 |
R | TCTGACAGTGCATCATCGCT |
Il1b
| F | CCTCGTCCTAAGTCACTCGC | 156 |
R | GCAGAGTCTTTTTGACCCTCCT |
Tnfa
| F | CTCTTCTCATTCCCGCTCGT | 104 |
R | GGGAGCCCATTTGGGAACTT |
MuRF1
| F | TGCAAGGAACACGAAGACGA | 170 |
R | ACAAGGAGCAAGTAGGCACC |
Atrogin1
| F | GGTCTCACGATCACCGACCT | 136 |
R | TCCACAGTAGCCGGTCTTCA |
Gapdh
| F | TGC CAC TCA GAA GAC TGT GG | 129 |
R | TTCAGCTCTGGGATGACCTT |