Lentix qrt pcr titration kits

Expression vectors pMIF-cGFP-zeo (System Biosciences, Palo Alto, CA) encoding miR-128a, and pmiR-ZIP-shRNA encoding miR-128a antisense oligonucleotides (miR-128a-AS) (AM11746; Applied Biosystems Inc, Foster City, CA) were constructed, respectively. Expression vectors and pPACKF1 vectors were transferred into 293T cells. Lentiviral particle titers (infectious units/ml) were quantified using LentiX qRT-PCR Titration Kits (Clontech Laboratories Inc, San Francisco, CA) following enrichment at 1,000,000×g for 60 min at 4 °C. Ten μl of aliquots of lentiviral particle mixtures containing 1 × 10⁹ infectious units/ml were then prepared for intra-articular injection.

Lentivirus-shuttled pMIF-cGFP-zeo expression vectors (System Biosciences) that coded the miR-29a precursor were constructed and co-transfected with pPACKF1 vectors into 293 T cells. After titration using LentiX qRT-PCR Titration kits (Clontech), each 10-μl suspension containing 1 × 10¹⁰ infectious units of lentivirus particles was prepared for injection into affected joints. Sixty-four male FVB/N mice were divided into control, OA, OA-miR-29a, and OA-mock groups. Injured joints (2 weeks after collagenase aggravation) were administered saline, lentivirus-shuttled miR-29a, and mock through an intra-articular injection. After euthanasia, 16 affected joints in each group were dissected for study at 8 weeks after collagenase injection. 8 injured joints were subjected to total RNA extraction and 8 were harvested for histologic analysis.

Animal use protocols were reviewed and approved by IACUC, Kaohsiung Chang Gung Memorial Hospital (IACUC Affidavit No. 2011062101). HEK293 cells were transferred with 1 μg pMIF-cGFP-zeo plasmid coding RPTOR along with 1 μg pPACKF1 plasmid (System Biosciences, Palo Alto, CA). Culture supernatants were harvested, ultrahigh centrifuged, and titrated using LentiX qRT-PCR Titration Kits (Clontech Laboratories Inc, San Francisco, CA) to prepare 2 × 10⁹ infectious units/ml lentivirus particle suspension. Twelve-week-old male FVB mice were anesthetized through inhale isoflurane. Twenty-four mice were evenly divided into two groups and anesthetized to receive 50 μl RPTOR or mock lentivirus particle suspension through tail vein injection. One week after administration, mice were intra-peritoneally injected with 10 mg/kg/day methylprednisolone for 4 weeks. For control group, 12 mice were injected with normal saline only. At the end of experiment, mice were killed to dissect femurs and tibiae for study. Six mice were used for μCT and histological assay, and 6 animals for harvesting bone extract for immunoblotting.