Fastgene scriptase 2 cdna synthesis kit

For quantitative RT-PCR, brains were dissected from 24 h starved or non-starved Canton-S male flies (n = 20) and total RNA was extracted using Total RNA Purification Plus Kit (Norgen Biotek). 1 ng-1 μg RNA was reverse transcribed to cDNA using FastGene Scriptase II cDNA synthesis kit (Nippon Genetics) using oligo dT primers in accordance with the manufacturer’s protocol. SensiFAST SYBR Hi-ROX Kit (Bioline) and primers targeting Tβh or tdc2 were used to perform quantitative real-time PCR on ABI PRISM 7000 Sequence Detection System. Each experiment was performed in technical triplicates. Rp15 was used as an internal control for normalization. Table S1 lists the sequence of primers used.

Isogen II was purchased from Nippon Gene Co., Ltd. (Tokyo, Japan). The FastGene Scriptase II cDNA Synthesis kit was purchased from NIPPON Genetics Co., Ltd. (Tokyo, Japan). DMSO, Dulbecco’s modified Eagle’s medium (DMEM; high glucose) without L-glutamine and phenol red, L-glutamine, LPS from E. coli O111, MEM nonessential amino acids (NEAA), Eagle’s minimum essential medium (EMEM) with L-glutamine and phenol red, N-1-Naphthylethylenediamine dihydrochloride, penicillin-streptomycin solution, phosphoric acid, and sulfanilamide were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Biowest (Nuaille, France).

Total RNA was extracted using the hot acid-phenol method (37 ). Extracted RNA was first subjected to DNase I (1 unit) treatment (Thermo Scientific) at 37°C for 30 mins, followed by incubation with 50 mM EDTA at 65°C for 10 mins. First-strand cDNA was synthesized using Fast Gene Scriptase II cDNA synthesis kit (Nippon Genetics) from 50 ng of total RNA according to the manufacturer's instructions. Real-time PCR quantification was performed using an ABI PRISM™ 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. PCR reactions were performed in 96 well plates using qPCRBIO SyGreen Blue Mix Hi-ROX (PCR Biosystems). The 2-ΔCT method was used to calculate the relative levels of expression of the target transcripts and normalised to Rpl32 mRNA or 18S rRNA. To inhibit transcription, the cells were cultured to OD₆₀₀ (∼0.7) and treated with 150 μg/ml 1,10-phenanthroline (Sigma). Cultures were removed at different time points (0, 2, 10, 20, 40 and 60 minutes) after addition of the drug, and immediately transferred to falcon tubes containing 40 ml of ice-cold water prior to RNA extraction.