Silencer sirna labeling kit

Primacy NPCs were isolated from human intervertebral disc (IDD patients and controls), mouse intervertebral disc (miR-150-5p KO and C57BL/6 wild-type mice). NPCs were preserved as a monolayer in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FCS), 100 IU/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% carbon dioxide at 37 °C. All the experiments involved the use of human NPCs at a confluence rate of 85%. As previously described [10 (link)], the cells were inoculated into 96-well plates for three-dimensional culture of the NPCs. The NPCs were then cultured at 37 °C and 5% carbon dioxide in 200 µl of NPC growth medium. After 21 days of culture, the cells were collected for further evaluation. Using a Silencer® siRNA labeling kit or miRNA negative control (mirVana miRNA mimics/inhibitor negative control #1) (Life Technologies), Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA), and Cy3-labeled or unlabeled miR-150-5p (a mirVana miRNA mimic or inhibitor) were transfected into human NPCs, SW1353 cells, or C28/I2 cells at 50 nM. The FBXW11 expression plasmid (pcDNA 3.1/V5-His TOPO TA Expression Kit) (Invitrogen) was subsequently obtained.

The cultured primary human NP cells were transfected with mimics or inhibitor labeled or unlabeled with Cy3 using the Silencer® siRNA Labeling Kit (#AM1636) or miRNA negative control (mirVanaTM miRNA mimics/inhibitor Negative Control #1, catalog number: 4464061/4464079) (Life Technologies) at 50 nM using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). To suppress SIRT1 expression, cells were transfected with either SIRT1 siRNA or control scrambled siRNA (Thermo Scientific Dharmacon®) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. The SIRT1 expression plasmid (pcDNA™3.1/V5-His TOPO™ TA Expression Kit) was obtained (Invitrogen™). At 48 h after transfection, the cellular lysates were collected to analyze the expression of genes of interest.

Synthetic miRNA mimics, anti-miR, and negative control miRNAs were purchased from ThermoFisher Scientific, and then labeled when needed and transfected as recommended^{8 (link), 62 (link)}. Briefly, synthetic miRNA were left unlabeled or labeled with Cy3 using the Silencer® siRNA labeling kit (#AM1632) from Life Technologies. For labeling, miRNA142-3p was incubated for 1 hour at 37 °C in the dark, and then precipitated with ethanol. 100 pmoles of unlabeled/labeled miRNA or anti-miR were transfected into cells grown in 10-cm dishes and cells were incubated for 24 h (in the case of HEK 293T cells) or 48 h in the case of astrocytes that were later co-cultured with LUAD cells at 5:1 ratio and utilized for imaging. For TRPA1 3’-UTR luciferase reporter assay, cells with miRNA mimics (ThermoFisher Scientific) and MISSION(R) 3’UTR Lenti GoClone (TM) TRPA1 (HUTR10124) from Sigma Aldrich were used to measure luciferase reporter activity with Mission Lightswitch Luciferase Assay (#MLS0001) from Sigma Aldrich according to manufacturer’s protocol, with 3 biological replicates each with 3 technical replicates.