Low rna input linear amplification labelling kit

Hepatocytes were isolated from perfused livers of two PNPASE (Pnpt1) liver-specific knockout C57BL/6 mice (HepKO; one male aged 12.9 weeks, one female aged 4.29 weeks, two independent experiments) and two sex-matched wild-type littermate mice. Total RNA was extracted from hepatocytes using TriZol Reagent (Invitrogen cat. #15596026) and the Qiagen RNeasy Mini Kit (Qiagen, cat. #74104). Labelled complementary RNA was generated using 200 ng of total RNA from each sample and the Agilent Low RNA Input Linear Amplification labelling kit. Each labelled sample was hybridized against its gender-matched sample in fluor-reversed pairs of arrays to an Agilent 4×44k Mouse Whole Genome Microarray. The arrays were scanned using the Agilent DNA Microarray Scanner, and data were extracted using the Agilent Feature Extraction (v.9.5.1.1) software using the standard Agilent protocol except without Lowess normalization. The fluor-reversed pairs were combined into Experiments in Rosetta Resolver 7.1 to produce the male and female signature gene ratios. We performed age and gender-matched differential expression analysis and generated a list of signature genes with significant differential expression in both the male and female cohorts. Both male and female cohorts showed very similar results. For simplicity, only fold expression changes in female mice were shown.