Transcripter first strand cdna synthesis kit

Total RNA was isolated from BV-2 cells with NucleoSpin RNA XS (74902, Takara Bio Inc. Kusatsu, Japan) according to the manufacturer’s instructions. Total RNA (0.5 μg) was reverse-transcribed into cDNA using a transcripter first strand cDNA synthesis kit (4379012, Roche). Messenger RNA (mRNA) abundance was determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) with LightCycler 480 SYBR Green I master (4707516, Roche) and specific primer sets. Data were collected and quantitatively analyzed with LightCycler 480 real-time PCR system (Roche) (95 °C; 10 s, 55 °C; 22 s, 72 °C; 5 s, 45 cycles). Gene expression was assessed by normalizing to β-actin. The PCR primers used in this study are listed below: forward strand IL-1β, 5′-AGTTGACGGACCCCAAAAG-3′; reverse strand IL-1β, 5′-AGCTGGATGCTCTCATCAGG-3′; forward strand β-actin, 5′-CTAAGGCCAACCGTGAAAAG-3′; reverse strand β-actin, 5′-ACCAGAGGCATACAGGGACA-3′.

RNA was isolated from E12.5 Pet-1^+/− and Pet-1^−/− animals from the rostral serotonin system using PureLinkTM RNA Mini Kit (Ambion by Life Technologies, Carlsbad, California). Purified RNA was converted to cDNA by PCR using the Transcripter FirstStrand cDNA Synthesis Kit (Roche Diagnostics, Indianapolis, Indiana) and stored at −20°C. qPCR was performed in triplicate using Fast StartTM Universal Syber Green ROX Master solution (Roche Diagnostics, Indianapolis, Indiana). Samples were normalized to β-actin.