Computational protocol: Upregulation of fatty acid synthesis and the suppression of hepatic triglyceride lipase as a direct cause of hereditary postprandial hypertriglyceridemia in rabbits

Similar protocols

Protocol publication

[…] The cDNA was synthesized from 1 µg of total RNA using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) and was subjected to PCR amplification using a PCR thermal cycler (ASTEC, model PC707, Japan). Primer sequences were designed based on the published data on rabbit genes and were used for RT-PCR and real-time RT-PCR. In case no proper primers were found in the literature, they were designed by using Primer3() or Primer-BLAST (NCBI, GenBank, BLAST). All PCR primers were designed to span at least one intron to distinguish PCR products generated from cDNA versus genomic DNA. These primer sequences together with sizes of the amplified fragments are shown in supplemental Table . The reaction mixture contained 0.1 µl of KOD Dash, 1.0 µl of 10 × PCR buffer, 1.0 µl of forward primer, 1 µl of reverse primer, 1.0 µl of cDNA template, and 4.9 µl of RNase- and DNase-free water (Toyobo). Thirty cycles of denaturation (98°C for 10 s), annealing (55°C for 2 s), and extension (74°C for 30 s) were carried out in the thermal cycler. The PCR products were separated via 1.5% agarose (Ultra pure agarose, Invitrogen) gel electrophoresis and visualized by staining with ethidium bromide under UV light illumination. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcript was used as a reference for RNA integrity and assay conditions. The quantitative real-time RT-PCR was carried out using an Applied Biosystems 7500 Fast Sequence Detection System, and was performed in duplicate using a SYBR Green Master Mix (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s protocol. Rabbit 18-S rRNA was amplified as an endogenous control to normalize input RNA. The amplification conditions included 10 min at 95°C followed by 40 cycles of 5 s at 95°C and 20 s at 60°C. The melting curve protocol was performed for each primer set to confirm specificity. […]

Pipeline specifications

Software tools Primer3, Primer-BLAST
Application qPCR
Organisms Oryctolagus cuniculus
Diseases Metabolic Diseases, Hypertriglyceridemia, Genetic Diseases, Inborn, Obesity, Abdominal