Illustra rnaspin midi rna isolation kit

Total RNA was extracted using the Illustra RNAspin Midi RNA isolation kit (GE Healthcare) and quantified spectroscopically with a Nanodrop 2000^® (Thermo Scientific). cDNA was synthesised using Transcriptor First Strand cDNA Synthesis Kit (Roche), which was performed in accordance with the manufacturer's protocol. Relative quantification of LPHN1 mRNA was performed using SYBR Green I Master reaction mix (Roche) and a LightCycler 480 (Roche). The house-keeping gene β-actin was used as a reference gene. The following primers were used at a final concentration of 0.5 μM: LPHN1, 5′-AGCCGCCCCGAGGCCGGAACCTA-3′ and 5′-AGG TTGGCCCCGCTGGCATAGAGGGAGTC-3′; Actin, 5′-T TCGCGGGCGACGATGC-3′ and 5′-GGGGCCACACGC AGCTCATT-3′.
PCR reactions began with incubation at 95°C for 3 min 30 s, then proceeded for 45 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 10 s. Fluorescence level was detected at 80°C in each cycle. A final elongation step was held at 72°C for 5 min. Raw fluorescence data were analysed using LinRegPCR quantitative PCR data analysis programme [25 (link)]. Amplified products were examined on 1.5% agarose gel containing ethidium bromide.

RNA was extracted using the Illustra RNAspin Midi RNA Isolation Kit (GE Healthcare, United States) and according to the protocol provided by the manufacturer. A two-step protocol was applied for qRT-PCR and the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems) was firstly used to convert RNA in cDNA. Thus, Power SYBR Green PCR Master Mix (Applied Biosystems) was used for relative RNA quantification. To this purpose, primers reported in Table 2 were used, and hrdB was chosen as internal standard. Two biological replicates and three technical replicates were used for each condition.