U6 small nuclear rna

Total RNA was extracted from frozen tissues or cultured cells using TRIzol Reagent (Thermo Fisher Scientific, St. Waltham, MA, USA) following the manufacturer's suggestions. MiR-205 expression was determined using TaqMan miRNA assays (Thermo Fisher Scientific) following the manufacturer's protocol, and U6 small nuclear RNA (GeneCopoeia, Carlsbad, CA, USA) was used as an endogenous control. The expression of IGF1R was determined using SYBR Green qPCR assay (TaKaRa, Dalian, People's Republic of China) and normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GeneCopoeia). The primers used in this study are as follows-IGF1R primers: 5′-GGACTACTACGCCAAAGAAG-3′ (forward) and 5′-TCAAAAGACAGCCACTCAGG-3′ (reverse); GAPDH primers: 5′-TTGGTATCGTGGAAGGACTCA-3′ (forward) and 5′-TGTCATCATATTTGGCAGGTT-3′ (reverse); miR-205 primers: 5′-CTTGTCCTTCATTCCACCGGA-3′ (forward) and 5′-TGCCGCCTGAACTTCACTCC-3′ (reverse); and U6 primers: 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse).