Effects of a transient lack of dietary mineral phosphorus on renal gene expression and plasma metabolites in two high-yielding laying hen strains
Effects of a transient lack of dietary mineral phosphorus on renal gene expression and plasma metabolites in two high-yielding laying hen strains
The animal trial was performed at the Agricultural Experimental Station of the University of Hohenheim, Germany. The experimental protocol is in strict compliance with the German Animal Welfare Legislation and approved by the Regierungspräsidium Tubingen, Germany, and the Animal Welfare Committee of the University of Hohenheim (Project no. HOH67-21TEr). The hens were fed the experimental diets for a period of four weeks before slaughtering in week 19 (n = 40) and week 24 (n = 40), respectively. Kidney samples (n = 80) were dissected, snap-frozen in liquid nitrogen and stored in -80 °C. Before RNA extraction, whole frozen kidney samples were ground with pestle and mortar while submerged in liquid nitrogen. About forty grams of each kidney sample were used for RNA isolation using TRI reagent (Sigma-Aldrich, Taufkirchen, Germany) and phenol-chloroform-based RNA extraction according to the TRI reagent protocol. To ensure the absence of DNA, the isolated RNA was treated with DNase I (Roche Diagnostics, Mannheim, Germany) and finally purified using the NucleoSpin RNA extraction kit (Macherey-Nagel, Düren, Germany). The RNA concentration was determined through spectrophotometry using the NanoDrop ND-2000 (Peqlab, Erlangen, Germany) and RNA quality of final RNA was analyzed with the Bioanalyzer 2100 device (Agilent Technologies, Waldbronn, Germany).  The range of RNA integrity numbers (RIN) was between 6.4 and 8.9, with an average of 7.8. The absence of genomic DNA contamination was confirmed with polymerase chain reaction (PCR) specific for a chicken GAPDH fragment. RNA libraries were prepared using the Illumina Stranded mRNA preparation kit (Illumina, San Diego, CA, USA).