Quantitative RT-PCR for BDNF Isoforms

Total RNA from cells was purified with RNeasy Micro kit (Quiagen) as recommended by the manufacturer and treated with DNase I using DNA-free kit (Ambion). First-strand cDNA was synthesised from 5 μg of total RNA with Superscript III reverse transcriptase (Life Sciences) according to manufacturer’s recommendations. PCR reactions were performed with HotFire polymerase (Solis Biodyne) in a volume of 10 μl containing 1/80 of reverse transcription reaction as a template. Human BDNF 5′ exons’ specific primers have been described previously [12 (link)]. Rat BDNF 5′ exons’ specific primers have been described previously [11 (link)] and were used in combination with hRluc specific antisense primer 5′-GTA CTT GTA GTG ATC CAG GAG GCG AT-3′.