Lightcycler faststart dna master hybridization probe kit

Total RNA from rat liver and FLC-5 cells was extracted with ISOGEN (Wako Pure Chem. Ind., Co., Ltd., Osaka, Japan) and the RNeasy Mini kit (Qiagen, Hilden, Germany), respectively. Gene expression in the RNA samples was evaluated by quantitative real-time PCR. First-strand cDNA was synthesized by reverse transcription (RT) using Omniscript (Qiagen). Subsequently, 2 µl of each RT reaction mixture was analyzed by LightCycler PCR (F. Hoffmann-La Roche Ltd. Diagnostics, Basel, Switzerland) using the LightCycler-FastStart DNA Master Hybridization Probe Kit or the FastStart DNA Master SYBR Green I Kit (Roche) according to the manufacturer’s protocols. The oligonucleotide primer sequences and accession numbers for target genes are shown in Table 1. Expression of both human and rat α-TTP and β-actin, and rat CYP4F2 were analyzed using the LightCycler-FastStart DNA Master Hybridization Probe Kit, and the expression of rat glutathione peroxidase (GPx) and ABCA1 was analyzed using the FastStart DNA Master SYBR Green I Kit. Expression data for target genes were normalized to β-actin copy number to compensate for differences in RT efficacy among samples as previously described.^{(18 (link))}

Total RNA from the liver was prepared using ISOGEN (Wako Pure Chem. Ind., Ltd., Osaka, Japan) according to the manufacturer’s protocol. Quantitative real-time PCR was performed to determine mRNA levels in the samples. Reverse transcription (RT) reactions were performed with Omniscript (Qiagen, Hilden, Germany). Subsequently, one-tenth (2 µl) of each RT reaction mixture was amplified by PCR using a LightCycler (F. Hoffmann-La Roche Ltd. Diagnostics Division, Basel, Switzerland) with the LightCycler FastStart DNA Master Hybridization Probe Kit or the FastStart DNA Master SYBR Green I Kit (F. Hoffmann-La Roche Ltd. Diagnostics Division) according to the manufacturer’s instructions. Real-time PCR was performed using oligonucleotide primers for BCMO, CYP26A1, LRAT, and β-actin, as previously described.^{(13 (link))} The oligonucleotide primers and accession number of genes are listed in Table 1.