RT-qPCR analysis of caspase-3, Bcl-2 and Bax mRNA expression

Total RNA was extracted from the thoracic aorta using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized with a First-Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). qPCR was carried out using a Maxima SYBR-Green PCR kit (Thermo Fisher Scientific, Inc.) with primers as listed in Table I. Following an initial 10 min at 95°C, the PCR thermal cycling program was performed as follows: 95°C for 15 sec, 60°C for 30 sec, and extension at 72°C for 30 sec, for 40 cycles. At the end of the reaction, melting curve analysis was performed to ensure the specificity of the reaction. Relative gene expression levels were determined using the 2^−ΔΔCq method (11,12). β-actin was used as an internal control.

Sequences of the oligo nucleotide primers used for reverse transcription-quantitative polymerase chain reaction analysis.

Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-like protein 4; bp, base pair.