Real-time quantitative PCR analysis of the effects of chronic ethanol feeding and partial hepatectomy on gene expression in single rat hepatocytes

Heaptocytes from four different rats were analyzed for gene expression. Two animals received alcohol containing liquid diet and two other littermates were pair-fed liquid carbohydrate diet for 6-8 weeks following the Lieber-De Carli feeding protocol. The rats were then anesthetized and subjected to 70% partial hepatectomy (PHx). The left lateral and medial (LLM) lobes were removed and quickly frozen in OCT. 24 hours post hepatectomy, the rats were anesthetized again and the remnant liver was harvested. Single hepatocytes were collected from LLM and PHx liver slices using laser capture microdissection. We used the 96.96 BioMark Dynamic Array (Fluidigm) to measure the expression of functionally relevant genes in each hepatocyte. RT-PCR gene expression profiling of 124 hepatocytes from livers of rats fed control diet (69 hepatocytes at 0 hour after resection (LLM) and 55 hepatocytes 24 hours after resection (PHx)) and ethanol diet (55 hepatocytes at 0 hour after resection (LLM) and 54 hepatocytes 24 hours after resection (PHx)). Entire cell lysate was used for gene expression analysis.