Dataset features


Application: Gene expression microarray analysis
Number of samples: 12
Release date: Aug 11 2010
Last update date: Mar 22 2012
Access: Public
Chemicals: Amino Acids, Glucose, Glycerol
Dataset link Identification and expression analysis of differentially expressed genes in a hepatocyte model of cold-induced glycerol production in rainbow smelt (Osmerus mordax)

Experimental Protocol

Rainbow smelt were collected by seine netting from Mount Arlington Heights, Placentia Bay, Newfoundland in late October 2007 and transported to the Ocean Sciences Centre, Memorial University of Newfoundland. The fish were held in a 3000 L indoor free-flowing seawater tank maintained at 8°C to 10°C and followed a natural photoperiod with fluorescent lights set by an outdoor photocell. Hepatocyte isolations were performed for individual male fish (i.e. no pooling of cells from different fish) from Nov. 27th to Dec.17th, 2007. Hepatocytes were not pooled as individual smelt produce different amounts of glycerol in response to cold temperature challenge. Each liver was perfused with medium containing collagenase and yielded approximately 300 million cells per individual. Initial or “pre-incubation” samples were collected and the remaining cell suspension was divided into 20 ml glass scintillation vials (6 x106 cells per vial) containing 2 ml of incubation medium and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature. Duplicate vials from each temperature were sampled at 24, 48 and 72 h incubation times for RNA isolation. RNA isolated from the hepatocytes of the 9 fish with the highest levels of glycerol production at 0.4°C were used to generate 2 mRNA pools (a “cold” pool and a “warm” pool) for each incubation time (24, 48 and 72 h). To summarize, the “cold” pools contained cells from 9 individual fish incubated at 0.4°C for either 24, 48 or 72 h and which exhibited the highest increase in glycerol levels at 72 h relative to pre-incubation levels, and the “warm” pools contained cells from these same 9 individual fish but incubated at 8°C for either 24, 48 or 72 h and therefore exhibited no increase in glycerol levels at 72 h relative to pre-incubation levels. Comparisons were made for the cold compared to warm pools at the 24, 48 and 72 h time points. For each time point, technical quadruplicate slides including two dye swaps were run per comparison. Incubation time: 24 h: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Incubation time: 48 h: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Incubation time: 72 h: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4 Technical replicate: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Technical replicate: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Technical replicate: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4










Jennifer Hall
Jennifer Rejeanne Hall