Enturbo sybr green pcr supermix kit

Total RNA was isolated with TRIpure Total RNA Extraction Reagent (ELK Biotechnology) and reverse transcription was conducted using EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology). After that, cDNA was subjected to qPCR using the EnTurbo™ SYBR Green PCR SuperMix kit (ELK Biotechnology) and analyzed with the StepOne™ Real-Time PCR System. The amplification conditions were as follows: 95°C for 3 min, 40 cycles of 95˚C for 10 sec, 58°C for 30 sec and 72°C for 30 sec. β-actin was employed as normalization controls. The 2^−ΔΔCt method was utilized for quantitation of gene expression. BCL6, forward: 5’- GCCCTATCCCTGTGAAATCTG-3’; reverse: 5’-GACGAAAGCATCAACACTCCAT-3’. av integrins, forward: 5’- GCTGGAACTCAACTCTTAGCTGG-3’; reverse: 5’- AGATGTGCTGAACAACTGGCC-3’. β3 integrins, forward: 5’- CTGTCCCTCATCCATAGCACC-3’; reverse: 5’- TAGAAGAACAGGCCACACGTG-3’. Actin, forward: 5’- GTCCACCGCAAATGCTTCTA-3’; reverse: 5’-TGCTGTCACCTTCACCGTTC-3’.

Total RNA was extracted using TRIpure Total RNA Extraction Reagent (ELK Biotechnology), and real-time PCR with three replicate wells per sample was performed on a StepOne™ Real-Time PCR machine (Life Technologies), using an EnTurbo™ SYBR Green PCR SuperMix kit (ELK Biotechnology, EQ001). Real-time quantitative PCR was performed in triplicate. The Ct values obtained from different samples were compared using the ΔΔCt method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal reference gene.

TRIpure Reagent (ELK Biotechnology) was used to extract the RNA from cells, and the EntiLink 1st Strand cDNA Synthesis Kit (ELK Biotechnology) was used to reverse-transcribe RNA into cDNA. Next, qPCR was conducted on the StepOne Real-Time PCR System using the EnTurbo SYBR Green PCR SuperMix Kit (ELK Biotechnology). U6 served as an internal control for miR-200b-3p. β-actin served as an internal control for HMGB3. The 2^−ΔΔCq method was used for the quantification of data. U6, forward 5′-CTCGCTTCGGCAGCACAT-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′; miR-200b-3p, forward 5′-GGCCCTAATACTGCCTGGTA-3′, reverse 5′-CTCAACTGGTGTCGTGGAGTC-3′; β-actin, forward 5′-GTCCACCGCAAATGCTTCTA-3′, reverse 5′-TGCTGTCACCTTCACCGTTC-3′; HMGB3, forward 5′-CTATGATCGGGAAATGAAGGATTAT-3′, reverse 5′-TTCTCATACTTCTCCTTCAGCTTTG-3′.

DOX was provided by Beijing Huafeng United Technology (Beijing, China). Antibodies against GAPDH, ATM, p-ATM, H2AX, p-H2AX, YES1, p-YES1, p53, p-p53, AMPKα, p-AMPKα were purchased from Abcam (Cambridge, UK). Dulbecco’s modified Eagle’s high glucose medium and Dulbecco’s modified Eagle’s medium were provided by GIBCO (Grand Island, New York, NY, USA). Dual-Luciferase Reporter Assay System Kit (Thermo Fisher Scientific, Waltham, MA, USA) and pSI-CHECK2 vector were provided by Hanbio Biotechnology (Wuhan, China). EntiLink™ 1st Strand cDNA Synthesis Kit and EnTurbo™ SYBR Green PCR SuperMix Kit were purchased from ELK Biotechnology (Wuhan, China).

TRIzol^® reagent (Thermo Fisher Scientific) was used to isolate total RNA from chondrocytes. To synthesize first-strand cDNA, an EntiLink^™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology) was used. Then, RT-qPCR was performed on a StepOne™ real-time PCR instrument (Life Technologies) with EnTurbo^™ SYBR Green PCR SuperMix Kit (ELK Biotechnology Co., Wuhan, China). Primer sequences were: β-actin, Forward: 5’-GTCCACCGCAAATGCTTCTA-3’, Reverse: 5’-TGCTGTCACCTTCACCGTTC-3’; hsa-circ-0134111, Forward: 5’-GAAAACAGATGAGGAGAAGGCC-3’, Reverse: 5’-CGTCTTTTTCTCAGCTTTGCC-3’; CCL1, Forward: 5’-CCAGATGTTGCTTCTCATTTGC-3’, Reverse: 5’-CAGGGCAGAAGGAATGGTGT-3’; U6, Forward: 5’-CTCGCTTCGGCAGCACAT-3’, Reverse: 5’-AACGCTTCACGAATTTGCGT-3’; hsa-miR-224-5p, Forward: 5’-CAAGTCACTAGTGGTTCCGTTTAG-3’, Reverse: 5’-CTCAACTGGTGTCGTGGAGTC-3’; miR-515-5p Forward: 5’-CGGGTTCTCCAAAAGAAAGCA-3’, Reverse: 5’-CAGCCACAAAAGAGCACAAT-3’. U6 was used as the internal control for hsa-miR-224-5p and miR-515-5p. The internal reference for the other genes was β-actin, and we used the 2^–ΔΔCT method for analysis of gene expression.

TRIzol (ELK Biotechnology, Wuhan, China) were used to extract total RNA from cells. Later on, complimentary DNA (cDNA) was synthesized from these extracted RNA using EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology). RT-qPCR was performed using EnTurbo™ SYBR Green PCR SuperMix Kit (ELK Biotechnology) and run on the StepOne™ Real-Time PCR (Life Technologies, Carlsbad, CA, USA).