Once the samples were isolated, they were stored in liquid nitrogen tanks unless we used them right away. Before adding Trizol, we take tissue samples from liquid nitrogen tanks and grind them with liquid nitrogen. Then, total RNAs were isolated from cartilage samples and chondrocytes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and were reversed transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Kyoto, Japan). cDNA synthesis was performed with 1 μg of total RNA utilizing oligo dt-primer with a cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Real time PCR was performed using TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Kyoto, Japan) on the qTOWER3G real time PCR system (analytikjena, Jena, Germany). Relative gene expression was calculated using ΔΔCt method and was normalized to U6 or GAPDH.25 (link)
The specific primer sequences (5’→3’) were as follows: miR-379-5p-F: GCGCTGGTAGACTATGGAA; miR-379-5p-R: GTGCAGGGTCCGAGGT, U6-F: CTCGCTTCGGCAGCACATATACT; U6-R: ACGCTTCACGAATTTGCGTGTCYBX1, YBX1-F: GGGTGCAGGAGAACAAGGTA; YBX1-R: TCTTCATTGCCGTCCTCTCT, PI3K-F: AGCATGGTCAGCTTTCTTCTT; PI3K-R: GAATGGAAGACGGGAGATTCA, AKT-F: CCAGGATCCATGGGTAGGA; AKT-R: GCAGCCCCTTTGACTTCTT, GAPDH-F: CCATGTTCGTCATGGGTGTGAACCA; GAPDH-R: GCCAGTAGAGGCAGGGATGATGTTCPI3K.
The specific primer sequences (5’→3’) were as follows: miR-379-5p-F: GCGCTGGTAGACTATGGAA; miR-379-5p-R: GTGCAGGGTCCGAGGT, U6-F: CTCGCTTCGGCAGCACATATACT; U6-R: ACGCTTCACGAATTTGCGTGTCYBX1, YBX1-F: GGGTGCAGGAGAACAAGGTA; YBX1-R: TCTTCATTGCCGTCCTCTCT, PI3K-F: AGCATGGTCAGCTTTCTTCTT; PI3K-R: GAATGGAAGACGGGAGATTCA, AKT-F: CCAGGATCCATGGGTAGGA; AKT-R: GCAGCCCCTTTGACTTCTT, GAPDH-F: CCATGTTCGTCATGGGTGTGAACCA; GAPDH-R: GCCAGTAGAGGCAGGGATGATGTTCPI3K.
