Avian myeloblastosis virus first strand cdna synthesis kit

Osteosarcoma and adjacent healthy tissue (weight, 25 mg) were harvested, and TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to extract total RNA. First-strand complementary (c)DNA was synthesized using the Avian Myeloblastosis Virus First-Strand cDNA Synthesis kit and oligo(dT) primers (Sangon Biotech Co., Ltd., Shanghai, China), according to the manufacturer's instructions. RT-qPCR was performed using LightCycler®480 software (Roche Diagnostics, Basel, Switzerland) with the SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). β-actin was used as the internal housekeeping gene and relative gene expression was calculated using the cycle threshold (Ct) method (2^−ΔΔCt). The PCR primers were as follows: Forward, 5′-AATAAAATCTTCCTGCCCACC-3′ and reverse, 5′-CTGTACTTGTCCGTCATGCTTC-3′ for CXCR4; forward, 5′-TGAGCACCTGTTTGCCTGAA-3′ and reverse, 5′-ATGAGCAGCACTCGGACCTT-3′ for β-catenin; and forward, 5′-TAGTTGCGTTACACCCTTTCTTG-3′ and reverse, 5′-TCACCTTCACCGTTCCAGTTT-3′ for β-actin. All experiments were independently performed in triplicate at least three times.