Thermal cycler cfx6 system

The extraction of total RNA from tissues and cells was implemented using Trizol reagent (Takara, Dalian, China) in accordance with the operation manual. Reverse transcription of lncRNA/mRNA and miRNA were conducted using a SuperScript Reverse Transcriptase Kit (Vazyme, Nanjing, China) and Poly (A) Tailing Kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Quantification of RNA was performed using SYBR Fast qPCR Mix (Thermo Fisher Scientific) in the Thermal Cycler CFX6 System (Bio-Rad, Hercules, CA, USA). The internal expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and endogenous small nuclear RNA U6 was used for the normalization of detected RNA using the 2^−ΔΔCt method, and the PCR program was: at 95°C for 10 min, followed by 95°C for 15 s and 60°C for 1 min for 40 cycles.
The sequences of primers were:

Total RNA was extracted from tissues and cultured cells using TRIzol reagent (Invitrogen, CA, USA) following the supplier’s directions. 1 mg of total RNA was converted to complementary DNA (cDNA) with specific primers in line with the protocol of a PrimeScript RT master mix kit (Takara, Tokyo, Japan). Quantitative real-time PCR was performed using a SYBR Green PCR kit (Takara) on Thermal Cycler CFX6 system (Bio-Rad, CA, USA). The amplification parameters were as follows: denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. The relative RNA expressions were calculated by the 2^−ΔΔCt method, normalizing to GAPDH or U6. A quantitative real-time PCR assay was performed at least three times.

Th overall RNA from the tissues and cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then subjected to reverse transcription using the PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan). The complementary RNA underwent a qRT-PCR analysis using the Thermal Cycler CFX6 System (Bio-Rad, Hercules, CA, USA), and the SYBR Green PCR Kit (TaKaRa). After normalizing miR-199a-5p to U6, the gene level was measured using the 2^-ΔΔCt method.

Total RNA from CDDP-resistant OS tissues or cells was extracted using an RNA extraction kit (Solarbio). The random primers (Solarbio) were used to perform reverse transcription, and the qRT-PCR was carried out on Thermal Cycler CFX6 System (Bio-Rad, California, USA) with SYBR mix (TaKaRa, Dalian, China). The amplification parameters were: denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30s, annealing at 60°C for 30s and extension at 72°C for 1min. The levels of OIP5-AS1 and FOSL2 were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the level of miR-377-3p was standardized via small nuclear RNA U6, then calculated with the method of 2^−ΔΔCt. The primer sequences were presented as follows: OIP5-AS1 (F, 5ʹ-TGCGAAGATGGCGGAGTAAG-3ʹ, R, 5ʹ-TAGTTCCTCTCCTCTGGCCG-3ʹ); miR-377-3p: (F, 5ʹ-GGGAGGCAGTGTATTGTTA-3ʹ, R, 5ʹ-CAGTGCGTGTCGTGGAGT-3ʹ); FOSL2 (F, 5ʹ-GAGAGGAACA AGCTGGCTGC-3ʹ, R, 5ʹ-GCTTCTCCTTCTCCTTCTGC-3ʹ); GAPDH (F, 5ʹ-TGTTCGTCATGGGTGTGAAC-3ʹ, R, 5ʹ-ATGGCATGGACTGTGGTCAT-3ʹ), and U6 (F, 5ʹ-ATTGGAACGATACAGAGAAGATT-3ʹ, and R, 5ʹ-GGAACGCTTCACGAATTTG-3ʹ).