7500 fast real time machine

After performing the KDM6A ChIP experiments for the T24, SV-HUC-1, and BdEC cell lines, qPCR was performed using the Roche FastStart Essential DNA Green Master (SYBR) kit on an Applied Biosystems 7500 Fast Real-Time machine, and the results are presented as % input IP. The experiments were performed as three technical replicates. The ChIP-qPCR results were calculated using the ΔΔct method. ChIP Ct values were normalized to the input.

cDNA was synthesized using qScript cDNA SuperMix (Quanta Biosciences) following the manufacturer’s protocol using the input fraction (total pituitary) or the immunoprecipitated fraction (gonadotrope) RNA. The synthesized cDNA was diluted 1:15, and 5 μL was used in each subsequent real time reaction using Fast SYBR Green Master Mix (Applied Biosystems) in a 20 μL final volume reaction on a 7500 Fast real time machine (Applied Biosystems). Cycling conditions were incubation at 95°C for 20 sec followed by 40 cycles of 95°C for 3 sec then 60°C for 30 sec followed by melt curve analysis. Primers were optimized for primer concentration, and primer efficiency was determined. Only primer sets with primer efficiencies between 95% and 105% were used. Primer sequences and concentrations are listed in Table 1. Rpl19 was used as the endogenous control and cDNA from LβT2 cells was used as the calibrator in the 2^-ΔΔCt calculation [37 (link)]. qPCR for each primer set was performed in duplicate for each sample isolated and the ΔCt mean used in calculations. 2^-ΔCt or 2^-ΔΔCt values were graphed for experiments performed on different days with the median with interquartile range shown. Although infrequent, amplicon values with high Ct values that exceeded the detectable range as determined by standard curve analysis were excluded. Thus “n” values ranged from 3 to 5 for any given primer set.

The RNA was obtained from skeletal muscle (gastrocnemius) with TRIzol (ThermoFischer^®, MA, USA) and submitted to cDNA conversion (High Capacity cDNA Reverse Transcription Kit; ThermoFischer^®, MA, USA). The quantitative Real-Time PCR was performed using the 7500 Fast Real-Time machine (Applied Biosystems^®, CA, USA) with SYBR Green PCR Mastermix (Applied Biosystems^®, CA, USA). The mRNA quantification for each gene was normalized with Gapdh using the 2^−ΔΔCT method. The primer sequences are listed in Table 1.