Cfx manager program version 3

Real-time PCR was conducted to detect RNA expression level of BDNF and miR-16 in hippocampal tissues. The Trizol (Invitrogen) extraction reagent, was used to extract hippocampal RNA and all procedures were carried out according to the manufacturer's instruction. Next, concentration and purity of the RNA samples were determined by Thermo NanoDrop 2000. One microgram of RNA from each sample was reverse transcripted into cDNA according to the instructions of the DBI-2220 Bestar™ qPCR RT Kit. Real-time PCR specific primers for rat miR-16, BDNF and β-actin (internal control) were designed using Primer Express software. The primers used in this study were as follows: miR-16 Forward: 5′-TAGCAGCACGTAAATTGGCG-3′;
Real-time PCR protocol was done following instructions from DBI-2044 Bestar® SybrGreen qPCR master mix. The reaction program for the PCR was as follows: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 5 s, 55°C for 30 s, 72°C for 30 s. All mRNA expression levels were detected by RT-PCR and detailed primer information can be seen in the Materials and Methods Section. Later the results were analyzed with the Biorad CFX manager program, version 3.0. Each experiment was done 3 times.

The RNA expression levels of NF-kB, iNOS, TNF-α, and IL-10 in the cortex (Table 1) were detected by using quantitative Real-time PCR. β-actin was utilized as an internal control, and all primers were adopted by Primer Express software. The RNA in cortex was extracted based on the instructions on the Trizol RNA (Beyotime Biotechnology, China) extraction kit. RNA concentration was measured with a Nanodrop 2000 ultraviolet spectrophotometer (Thermo Fisher, Wilmington, DE 19,810 USA). According to the procedure of the DBI-2220 qPCR reverse transcription kit (Bestar, China) total RNA was reverse transcribed into cDNA, and quantitatively analyzed using Bestar SYBR Green qPCR master mix. The reaction parameters were: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The quality was probed by melt curve. The Bio-Rad CFX manager program (version 3.0.) was used to analyze the data, while the 2−∆∆Cq method was used to monitor the relative expression level of gene. To ensure a good reproducibility of data, each experiment was triplicated.