Unformatted text preview: qPCR analysis was performed according to the manufacturer’s recommendations. Briefly, PAX3 or IgG immunoprecipitated DNA samples (IPs) and input DNA samples were loaded with RT2qPCR SYBR Green/Fluorescein Master Mix (Qiagen) into arrays and amplified for 40 cycles on iQ5 cycler (BioRad). Additionally, MITF, DCT and GAPDH genomic sites were amplified with KAPA SYBR FAST qPCR Master Mix (KapaBiosystems) for 40 cycles. Each PCR reaction was performed in duplicate andmean Ct values were used for quantification. Each IP Ct value was normalised 81 Chapter IV – Medic, Rizos and Ziman, 2011 against the Input Ct value for the same PCR assay (ΔCt), before the fold enrichment in PAX3‐IP over negative control IgG‐IP (ΔΔCt) was calculated for each genomic site. Enrichment for the non‐specific site (GAPDH) was determined to be the background and only enrichment above this value was considered true enrichment for each specific genomic site. Specific genomic enrichment was then calculated for each individual ChIP experiment, by subtracting the background from it, and the mean value for duplicate ChIP experiments was calculated. RNA extraction and RTqPCR Total RNA was extracted from cultured HEM1455 and A2058 cells using either Isolate RNA Mini Kit (Bioline), or Tryzol reagent (Invitrogen) with a column clean up (Qiagen). Quantity and quality of RNA were measured with a nanodrop spectrophotometer and assessed by agarose gel electrophoresis. 250ng of total RNA was reverse transcribed using Omniscript RT kit (Qiagen) and PCR products were amplified with either KAPA SYBR FAST qPCR Master Mix (KapaBiosystems) or SYBR GreenER qPCR SuperMix (Invitrogen), for 40 cycles. Each PCR reaction was performed in triplicate and the mean Ct value was used to calculate fold change over GAPDH (ΔΔCt). Primers used for RT‐qPCR are listed in the supplementary information (Supplementary Table S2). Statistical analyses Statistical analyses of specific site enrichment and gene expression were performed using a Student’s t‐test. 82 Chapter IV – Medic, Rizos and Ziman, 2011 Results PAX3 expression in...
View Full Document
- Spring '14