RNA extraction and real-time PCR
TRIzol (Invitrogen, USA) was used to extract total RNA from the
supernatant of the cells and the mouse hippocampal tissue. By using
NanoDrop 2000 micro-ultraviolet spectrophotometry (1011U, NanoDrop
Technologies, USA), the purity and concentration of RNA were determined.
Then the cDNA was synthesized according to the instructions provided
with the cDNA Reverse Transcription Kit (Takara, Japan). PCR was
conducted as follows: 95 °C for 30 seconds, followed by 40 cycles of 5
seconds at 9 5°C and 34 seconds at 60 °C. The expression of the target
genes was normalized relative to that of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), and calculated using the 2−ΔΔCt method (Ayuket al. , 2016). The primer sequences are: COX-2, forward: CAC TAC
ATC CTG ACC CAC TT, reverse: ATG CTC CTG CTT GAG TAT GT; TNF-a, forward:
GAT AAA GGG ACA GCG TCA GC, reverse: CAG CCT TGT CCC TTG AAG AGA ACC;
iNOS, forward: GAT AAA GGG ACA GCG TCA GC, reverse: CCT TCG GGC CAA AGA
TCC TG; IL-1β, forward: ATC TCG CAG CAG CAC ATC AAC, reverse: TGT TCA
TCT CGG AGC CTG TAGT; GAPDH, forward: GCC AAA TTC AAC GGC ACA GT,
reverse: AGA TGG TGA TGG GCT TCCC.