*2*.*7*. *Real-Time Polymerase Chain Reaction (PCR) Analysis*

The expression levels of SVCT2 (*Slc23a2*) and GLUT1 (*Slc2a1*) mRNA were assessed by quantitative real-time polymerase chain reaction (PCR) as described previously [32]. The rats subjected to MCAO were killed after 24 h of reperfusion, and total RNA samples were prepared from the ischemic penumbral cortex of each rat. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. Total RNA (500 pg) from each sample was reverse-transcribed with oligo-dT and random hexamer primers using reverse transcriptase (PrimeScript RT Enzyme Mix I; Takara RNA PCR Kit, Takara Biomedicals, Shiga, Japan). Real-time PCR was performed with 10 ng of cDNA and a pair of gene-specific primers (Takara Biomedicals, Shiga, Japan) that were added to the SYBR Premix EX *Taq* (Takara Biomedicals, Shiga, Japan) and subjected to PCR amplification on the iCycler iQ Real-Time Detection System (Bio-Rad Laboratories, Hercules, CA, USA) (1 cycle at 95 °C for 10 s and 50 cycles at 95 °C for 5 s and 60 °C for 34 s). β-Actin expression was used to normalize the cDNA levels. The PCR products were analyzed using a melting curve to ascertain specificity of the amplification. The data were expressed as mean ± SD relative to the sham-operated nondiabetic group.
