| Assay Method Information | |
| | Biochemical EC50 Assay |
| Description: | Cell culture: Cells were cultured in RPMI1640 medium+15% FBS. Cells were maintained at a density between 2e5/mL and 1e6/mL. Cells were centrifuged, resuspended in fresh medium, counted and plated at 150,000 cells per well in 100 uL in a non-coated, flat bottom tissue culture plate. Compound treatment: 10 mM stock solution of FA GeneTAC was diluted 1:10 in DMSO followed by a 1:100 dilution in growth medium. Working solution was then further diluted to 10× desired final concentration of 150 nM. Compound was then diluted at a 1:3 ratio into growth medium containing 0.01% DMSO. 5-point, 3-fold dose response curve was generated. 11 μL of 10× compound was added to wells containing 100 μL cell suspension of GM15850. 11 μL growth medium containing 0.01% DMSO was added to all wells not treated with FA GeneTAC. Cells were allowed to incubate for 48 hrs prior to cell lysis using guanidine isothiocyanate solution. RNA isolation: Total RNA was isolated and purified in 384-well column filter plates using chaotropic salt. qRT-PCR: qRT-PCR reactions were assembled using AgPath-ID reagents (Thermo Fisher) using 6 uL mastermix and 4 μL RNA. qRT-PCR TaqMan primer probe sets against human FXN (Assay ID Hs01075496_m1) and human GAPDH (Assay ID Hs00266705_g1) were used to measure the intended targets. qRT-PCR was run on the ThermoFisher QuantStudio 6 PRO instrument using the manufacturer's recommended cycling conditions. Data analysis: qPCR data was analyzed using Thermo Fisher Design and Analysis software. Data was exported to Excel and hFXN expression was normalized to hGAPDH expression. |
| Affinity data for this assay | |
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