Computational protocol: Effects of Feeding Milk Replacer Ad Libitum or in Restricted Amounts for the First Five Weeks of Life on the Growth, Metabolic Adaptation, and Immune Status of Newborn Calves

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Protocol publication

[…] The liver tissue was crushed to a fine powder under liquid nitrogen using a mortar and pestle while snap frozen. The powdered tissue (50–100 mg) was homogenized using a FastPrep FP120A-230 (Thermo Electron Corporation, Milford, MA) cell disrupter system to extract total RNA using Trizol Reagent (Life Technologies, Darmstadt, Germany). The contamination of the extracted RNA with genomic DNA was removed using RNase-Free DNase (Qiagen GmbH, Hilden, Germany). The samples were purified using the RNeasy Mini Kit (Qiagen GmbH). The integrity and quality of total RNA were confirmed upon gel electrophoresis on denaturing agarose gels stained with ethidium bromide and after measuring the optical density at 260:280, which was 1.85 to 1.9, using a spectrophotometer (NanoPhotometer; Implen GmbH, Munich, Germany). For cDNA synthesis, 1 μg of RNA was reverse-transcribed using 200 U of the Reverse Transcriptase MMLV-RT RNase (H-) Point Mutant (Promega Corporation, Madison, WI) and 250 pmol random hexamer primers (Metabion International AG, Planegg-Steinkirchen, Germany). The obtained cDNA was diluted 1:4 with diethylpyrocarbonate (DEPC) water, and the aliquots were stored at -80°C. Specific primers were used to measure the mRNA expression of IGF 1 (IGF1), growth hormone receptor (GHR), IGF binding protein 1 (IGFBP1), IGFBP-2, IGFBP-3, IGFBP-4, insulin-like growth factor 1 receptor (IGF1R), insulin receptor (INSR), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase, cytosolic isoform (PCK1) and mitochondrial isoform (PCK2), glucose-6-phosphatase (G6PC), propionyl-CoA carboxylase alpha chain, mitochondrial (PCCA), and solute carrier family 2, facilitated glucose transporter 2 (SLC2A2). The primers were designed using Primer-BLAST at NCBI (National Center for Biotechnology Information) or according to previous studies (), and real-time PCR was performed on a LightCycler 2.0 using FastStart DNA Master Plus SYBR Green I Master Mix (Roche Diagnostics GmbH, Mannheim, Germany) with 2 μL of cDNA. Each cDNA sample was analyzed in duplicate. To verify specific PCR products, a melting curve analysis was performed after the last amplification cycle. Furthermore, the product purity and size were confirmed using agarose gel electrophoresis, showing a single band at the expected size, followed by sequencing (ABI 3130 Genetic Analyzer (Life Technologies). The efficiency was calculated using LinRegPCR 2013 software []. Samples with an efficiency below 1.75 were discarded, and the preparation was repeated. The data calculation was performed using LightCycler analysis software 4.05 to evaluate the quantification cycles. The selection of appropriate reference genes and the quantification of the data were performed using qBase+ software (Biogazelle NV, Zwijnaarde, Belgium) []. Low-density lipoprotein receptor-related protein 10 (LRP10), emerin (EMD), and eukaryotic translation initiation factor 3 subunit K (EIF3K) served as reference genes.The glycogen and glucose concentrations in the liver were determined using an enzyme-based starch-kit (no. 10207748035; Roche Diagnostics GmbH, Mannheim, Germany) with wet tissue (25 mg) frozen under liquid nitrogen and then subjected to a mortar and pestle while snap frozen. […]

Pipeline specifications

Software tools Primer-BLAST, LinRegPCR, qbase+
Application qPCR
Organisms Bos taurus
Chemicals Glucose, Phosphoenolpyruvate, Triglycerides, Urea