Abi 7900ht sequence detection machine

Total RNAs were extracted from tissues by using Trizol (Invitrogen, USA) following the manufacturer's instruction as previously described.²¹ Reverse transcription was performed by using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (Yeason Biotechnology, Shanghai) to obtain cDNA. RT‐qPCR was performed by using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeason Biotechnology, Shanghai) on an ABI 7900HT sequence detection machine (Thermo Fisher Scientific, Massachusetts, USA). GAPDH RNA was used as an internal control. The 2^−ΔΔCT method was used to calculate different expression of RNAs. The experiments were repeated three times. All primers used were listed in Table S3.

Total RNA was isolated from tissue samples or cells using a Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The first-strand cDNA for miR-135a-5p, BMI1 and DANCR was synthesized from total RNA using MicroRNA Reverse Transcription Kit and TaqMan Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA), respectively. The first-strand cDNA was used to carry out qRT-PCR with 2× SYBR Green qPCR Mix (Aidlab, Beijing, China) on an ABI 7900HT sequence detection machine (Thermo Fisher Scientific). The primers used were presented as below: for DANCR, 5′-GCCACAGGAGCTAGAGCAGT-3′ (forward) and 5′-GCAGAGTATTCAGGGTAAGGGT-3′ (reverse); for miR-135a-5p, 5′-CCGGCGTATGGCTTTTTATTCC-3′ (forward) and 5′-CAGTGCAGGGTCCGAGGT-3′ (reverse); for BMI1 5′-ACTGGAAAGTGACTCTGGGA-3′ (forward) and 5′-TACTGGGGCTAGGCAAACAA-3′ (reverse); for GAPDH, 5′-TATGATGATATCAAGAGGGTAGT-3′ (forward) and 5′-TGTATCCAAACTCATTGTCATAC-3′ (reverse); for U6 (Forward, 5′-CTCGCTTCGGCAGCACATATACT-3′ (forward) and 5′-ACGCTTCACGAATTTGCGTGTC-3′ (reverse). GAPDH was deemed as a reference gene of BMI1 and DANCR, while U6 was an internal control of miR-135a-5p. The relative expression levels of miR-135a-5p, BMI1 and DANCR were evaluated with 2^−ΔΔCt method.

Total RNA was extracted from tissue samples and cells on ice using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA using the TRUEscript 1st Strand cDNA Synthesis Kit (Aidlab, Beijing, China) following the manufacturer’s protocol; reactions were incubated for 30 min at 42°C, 5 min at 85°C, and then stored at −20°C. qRT-PCR reactions were performed using 2×SYBR Green qPCR Mix (Aidlab) on an ABI 7900HT sequence detection machine (Thermo Fisher Scientific, Waltham, MA, USA); reactions were incubated at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 40 s. GAPDH was used as an internal control. The primer sequences were as follows: linc-ROR forward: 5'-GAAGGTTCAACATGGAAACTGG-3', and reverse: 5'-TGAGACCTGCTGATCCCATTC-3'; GAPDH forward: 5'-CTCAGACACCATGGGGAAGGTGA-3', and reverse: 5'-ATGATCTTGAGGCTGTTGTCATA-3'. Both target (linc-ROR) and reference (GAPDH) genes were amplified in separate wells and run in triplicate. Statistical analyses of the results were performed using the 2^−ΔΔCT relative quantification method.

We conducted RNA extraction using TRIzol reagent (Invitrogen) as previously described [33 (link)]. We obtained cDNA using a pTRUEscript 1^st Strand cDNA Synthesis Kit (Aidlab, Beijing, China). We conducted RT-qPCR with the 2× SYBR Green qPCR Mix (Aidlab) on the ABI 7900HT sequence detection machine (Thermo Fisher Scientific, Waltham, MA, USA). Expression fold differences were determined via the 2^−ΔΔCT method.