Oligo dt primer

TRIzol reagent (Takara, Dalian, China) was used to extract total RNA from the ovary. The isolated RNA was treated with DNase (Takara, Dalian, China), to remove potential genomic DNA contamination. First-strand cDNA was synthesized with M-MLV reverse-transcriptase (Takara, Dalian, China) and oligo (dT) primer (Sangon, Shanghai, China). The quality of RNA was assessed by formaldehyde agarose gel electrophoresis and was quantified spectrophotometrically. After pyriproxyfen exposure, the concentrations of RNA in control group and treatment group were more than 1000 ng/μL. The OD_260/280 in the control group and treatment group was 1.90–2.00 after pyriproxyfen exposure.

Total RNA from cells was extracted using the Trizol reagent, following by reverse transcription for purified cDNA templates. A SYBR Premix Ex Taq II Kit (Takara) was used to perform real-time RT-PCR in the presence of oligo dT primers (Sangon) according to the manufacturer’s recommendation. The mRNA expression was normalized to β-Actin.

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.

mRNA differential display (DD) was performed essentially as previously described [61 (link)] with the total RNA of RRIC52. First-strand cDNA was synthesized using 2 μg total RNA as template by the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Waltham, MA, USA) according to the manufacturer’s instruction. Three sets of anchored 3’-oligo (dT) primers (H-T11M, M = A/G/C) were used to synthesize first-strand cDNA. PCR amplification was performed using in combination with one reverse transcription step anchored primer and one arbitrary primer (B0301-B0326, Ap27-Ap34 in Supplementary Material, Table S4). The oligo (dT) primers and arbitrary primers were obtained from Sangon Biotech Corp (Shanghai, China). Each reaction mixture, in a total volume of 20 μL, contained 2 μL first strand cDNA, 2 mM each of dNTPs, 0.2 μM of random primers, 0.2 μM of anchored primer, and 1 unit of Taq DNA polymerase (Fermentas). The cycling parameters were as follows: 94 °C for 30 s, 40 °C for 2 min, 72 °C for 30 s for 40 cycles followed by 72 °C for 5 min. The amplified cDNAs were then separated on a 6% denaturing polyacrylamide gel.