Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from
tumor protein p53 was extracted using the RNeasy Mini Kit (Qiagen, Hilden Germany) according to the manufacturer’s directions. First-strand complementary DNA (cDNA) was synthesized with random primers and SuperScript II reverse transcriptase (Invitrogen). The RT reaction was carried out at 42 °C for 50 min, and then at 70 °C for 15 min. PCR amplification was performed by denaturation at 94 °C for 30 s, annealing at 58 °C or 62 °C for 1 min, and extension at 72 °C for 1 min and 45 s, using template cDNA and TaKaRa Ex Taq (Takara Bio, Shiga, Japan). PCR products were electrophoresed on 1.5% agarose gels and detected with ethidium-bromide staining. The sequences of the primers and product length were as follows: glyceraldehyde-3-phosphatate dehydrogenase (GAPDH) sense 5′-GGTGAAGGTCGGTGTGAACGGATTT-3′ and antisense 5′-AATGCCAAAGTTGTCATGGATGACC-3′ (478 bp), CX3CR1 sense 5′-TCATCACCGTCATCAGCATCG-3′ and antisense 5′-TTGAGGCAACAGTGGCTGAAC-3′ (511 bp), tartrate-resistant
acid phosphatase (TRAP) sense 5′-TCCCCTGGTATGTGCTGG-3′ and antisense 5′-GCATTTTGGGCTGCTGA-3′ (220 bp), CCR1 sense 5′-CTACAAGAGCCTGAAGCAGTG-3′ and antisense 5′-GGTACTTCCAGAACCGTTCAC-3’ (371 bp).
