First strand cdna kit

RNA was isolated using the RNAeasy kit (Qiagen), and cDNA synthesis was done using the first strand cDNA kit (ROCHE). qRT–PCR was performed on a LC480 Light Cycler (Roche) using SYBR Green (Roche) and the following primers: TSC1 5′‐CAA ACT CCA GGC AAG AGG AC‐3′ (forward) and 5′‐CCA ATT CAA ACA CCT GGG TTA‐3′ (reverse), TSC2 5′‐TGC AAG CCG TCT TCC ACA T‐3′ (forward) and 5′‐ATG GAC ACA AAG TCG TTG C‐3′, β‐actin 5′‐AGA GGG AAA TCG TGC GTG AC‐3′ (forward) and 5′‐CAA TAG TGA TGA CCT GGC CGT‐3′ (reverse), ATP5G1 5′‐AGG GCT AAA GCT GGG AGA CTG AA‐3′ (forward) and 5′‐GTC TGG CCA CCT GGA GTG GGA‐3′ (reverse), CYCS 5′‐AGG GAC AGA ATT TAA ATA TGG GTG A‐3′ (forward) and 5′‐AGA TTT GGC CCA GTC TTG TG‐3′ (reverse), HPRT (for normalization) 5′‐TGA CAC TGG CAA AAC AAT GCA‐3′ (forward) and 5′‐GGT CCT TTT CAC CAG CAA GCT‐3′ (reverse). For microRNA qRT–PCR, RNA was isolated using the miRNeasy kit (Qiagen), and primers obtained from the miScript System (Qiagen) were used for expression analysis with a primer for U6 RNA used for normalization. Primers for the analysis of mitochondrial DNA to genomic DNA ratio: genomic glucagon‐intron 5′‐TGA CAA AGA CGG ACT TGA CG‐3′ (forward) and 5′‐CCC TGT GTC ACA AGC AGA TG‐3′ (reverse); mitochondrial COX2 5′‐TTC ATG ATC ACG CCC TCA TA‐3′ (forward) and 5′‐TAA AGG ATG CGT AGG GAT GG‐3′ (reverse).