Uracil dna glycosylase

Tissue culture cell RNA were isolated using the RNAqueous kit whereas pancreatic and liver RNA were isolated using the ToTALLY RNA kit (Ambion, Carlsbad, CA). The Turbo DNA-free DNAse Treatment Kit (Ambion, Carlsbad, CA) was then used to remove trace genomic DNA followed by cDNA generation using the iScript DNA Synthesis Kit (Bio-Rad, Hercules, CA). Gene expression was then quantitated by PCR using the dUTP-containing FastStart SYBR Green Master Mix in conjunction with Uracil-DNA-Glycosylase (Roche, Nutley, NJ). Fold induction of gene expression was calculated using the 2(-ΔΔC(T)) method (Livak and Schmittgen 2001 (link)). PCR primer sequences are provided in Supplemental Material. The efficiency of primer amplification was tested in cDNA dilution analyses. These showed that correlation coefficients were ~0.99 and PCR efficiency was ~95%.

Tissue RNA was isolated using the ToTALLY RNA kit whereas islet RNA was isolated using the RNAqueous kit (Ambion, Carlsbad, CA). The Turbo DNA-free DNAse Treatment Kit (Ambion, Carlsbad, CA) was then used to remove trace genomic DNA followed by cDNA generation using the iScript DNA Synthesis Kit (Bio-Rad, Hercules, CA). Gene expression was then quantitated by PCR using the dUTP-containing FastStart SYBR Green Master Mix in conjunction with Uracil-DNA-Glycosylase (Roche, Nutley, NJ). Fold induction of gene expression was calculated using the 2(-ΔΔC(T)) method (Livak and Schmittgen 2001 (link)). PCR primer sequences are provided in Supplemental Material.