4.6. qRT-PCR to Detect BDNF and NT-3 mRNA Expression

To examine axonal outgrowth-related gene expression, the primary cortical neuron cells co-cultured with PLA and PLA/DHA CSNM for 3 days were subject to quantitative real-time polymerase chain reaction (qRT-PCR) analysis and compared with control runs using culture medium only. The expression of neural marker gene brain-derived neurotropic factor (BDNF) and neurotrophin-3 (NT-3) was analyzed using standard protocols for RNA isolation and cDNA synthesis. Total RNA was extracted from the primary cortical neuron cells using TRIzol^® reagent (Invitrogen) and reversely transcribed into cDNA by using Total RNA Isolation Kit and Maxime RT PreMix Kit according to the manufacturer’s protocols. The qRT-PCR was conducted using an SYBR Green RT-PCR kit in a MiniOpticon^TM real-time PCR detection system (Bio-Rad CFD-3120). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acted as the housekeeping gene, and relative mRNA expression of BDNF and NT-3 was determined using the 2−ΔΔct relative quantification method. The primers used are listed in Table 1.

Apoptotic primer sequence used for qRT-PCR analysis.

¹ NT-3, neurotrophin-3; ² BDNF, brain-derived neurotropic factor; ³ GAPDH, glyceraldehyde 3-phosphate dehydrogenase.