Lightcycler faststart dna master sybr green 1 pcr kit

To validate the microarray results, quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the Lightcycler-Faststart DNA master SYBR green I PCR kit (Roche, Basel, Switzerland) according to the manufacturer's specifications. The comparative threshold cycle (CT) method was used for the calculation of amplification fold. To verify the microRNA expression profile, stem-loop microRNA qRT-PCR was performed using the small RNA U6 for an internal control.[7 (link)] The relative quantity of each microRNA in each sample, normalized to U6 RNA and relative to the expression in control samples, was calculated using the comparative CT (ΔΔCT) method, with the equation of RQ = 2^−ΔΔCT, where ΔΔCT = (CT_microRNA − CT_U6) _Exp − (CT_microRNA − CT_U6) _Control

To detect the expression of APAF1, TOSO, and Caspase3, real-time quantitative RT-PCR was performed using the Lightcycler-Faststart DNA master SYBR green I PCR kit (Roche Applied Science, Indianapolis, IN) in a Roche Lightcycler 1.2 Real-Time PCR System (Roche) according to the manufacturer’s instructions. We used the following primers (forward/reverse, 5′-3′):
β-actin was used as an internal control. The comparative threshold cycle method was used for calculation of amplification fold. The expression level of each gene was normalized by dividing by the expression level of β-actin.