Computational protocol: Lactoferrin Promotes Early Neurodevelopment and Cognition in Postnatal Piglets by Upregulating the BDNF Signaling Pathway and Polysialylation

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Protocol publication

[…] Hippocampal tissues (~10–20 mg wet weight) were homogenized in a lysis buffer (Agencourt) purchased from Beckman Coulter, USA. Total RNA was prepared from each tissue sample using the Agencourt RNAdvance™ Tissue Kit (Beckman Coulter, USA) following the automated procedure described by the MICROLAB® STAR Liquid Handling Workstation (Hamilton Robotics GmbH Bonaduz GR, Switzerland) []. The quality of the RNA was monitored using an Agilent 2100 Bioanalyzer. Briefly, 250 ng of total RNA (integrity numbers >7.5) was used to generate double-stranded complementary DNA (cDNA), followed by in vitro transcription and complementary RNA (cRNA) labeling with biotin. Biotin-labeled cRNA (12.5 μg) was added to the hybridization mixture containing the control oligonucleotides, hybridization buffer, and water. A total of 18 hybridization mixtures, nine each for the Lf and control groups, were applied to a specialized Porcine Microarray Chip (Catalog No. 900625 GeneChip® Porcine Genome Array, Affymetrix). After hybridization for 16 h at 55 °C, the microarray chips were washed to remove nonhybridized material, stained with streptavidin, and then scanned with a GeneChip Scanner 3000 7G 4C w/7G Workstation. Signal intensities were determined and analyzed using the Affymetrix GeneChip Command Console software. All genes were imported into Partek Genomics Suite 6.5 software (Partek Inc., USA, www.partek.com) for one-way ANOVA analysis. Data were expressed as absolute intensities. Quality control of the data was carried out on all samples using a Pearson correlation matrix. No outliers were identified using the principal component analysis method with Partek software. After normalization, results were analyzed via log2, transformed, and analyzed by Student’s t test. Genes showing significant differences (p < 0.05) were selected and analyzed using the Ingenuity® System (Redwood City, CA, USA, http://www.ingenuity.com) and the Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/). […]

Pipeline specifications

Software tools Partek Genomics Suite, DAVID
Application Gene expression microarray analysis
Chemicals Iron, N-Acetylneuraminic Acid