1. Aim visit.
The STSM took place in une with to the Roslin Institute (UK) with the purpose to successfully finish our research on evaluation of the effectiveness of gamma- and alpha-tocopherol in case of dietary induced oxidative stress in chickens.
2. Work carried out
One part of the study included the identification of differences in gene expression in gamma- and alpha-tocopherol supplemented chickens. The microarray analyses have been conducted in collaboration with Centre for Functional genomics at Roslin Institute. Because we had no experiences in working with microarrays and analyzing the gene expression data and had no access to analysis software we needed knowledge and the experience of Roslin researchers in order to do the analyses and the interpretation of the data correctly, decrease the time of data evaluation and thus speed up the publication of the work.
Firstly we joined Alison Downing in the laboratory of ARK Genomics and witnessed the laboratory preparation of samples according to the standard protocol for Affymetrics chips. Since cDNA synthesis, labelling and hybridization require incubation times we were in the mean time learning about the hardware and software for the gene chip analysis and quality control.
In the next step we got the raw data of our experiment (laboratory analyses for these samples were done previously by the ARK genomics) and learned how to use Partek genomic suite for the statistical evaluation of the data. Once we got familiar with the software, we’ve analyzed the data and identified differentially expressed genes between dietary treatments. After the genes were identified we had to identify and connect the metabolic pathways they are involved in and find the key mechanistic differences among treatment groups. We created gene lists which were than later used for the upload into Ingenuity software for biological interpretation of data. When we got familiar with the Ingenuity software we’ve used it to identify relationships, mechanisms, functions, and pathways of relevance of genes which were differentially expressed among treatment groups. This was the work we spent most of the time on, since we needed to get the good biological interpretation and we also had to choose the key gens which would be than later verified with Real-time PCR method.
The evaluation of the data led us to some interesting results. The most important differences in gene expressions were between the groups fed different sources of fat (saturated vs. polyunsaturated fat) and between groups fed different isomers of vitamin E (alpha- and gamma-tocopherol). The most important genes which were up regulated in group fed saturated fat vs. group fed polyunsaturated fat were involved in lipid metabolism, while genes which were down regulated were involved in oxidation-reduction processes. Genes that were down regulated in group supplemented with alpha-tocopherol vs. group supplemented with gamma-tocopherol were involved in fatty acid metabolic processes, oxidation-reduction processes and cholesterol metabolism. This is a confirmation of results from previous studies on cell lines and in mammals that alpha-tocopherol down regulates cholesterol biosynthesis. It was shown for the first time that gamma-tocopherol does not possess the same ability. There was also an interesting difference between the group supplemented with gamma-tocopherol and the unsupplemented group. There were many genes involved in immune and inflammatory response that were down regulated in the gamma-tocopherol supplemented group compared to unsupplemented positive control. This effect was not observed with alpha-tocopherol supplementation.
After we returned from UK, we selected the genes for Real-time PCR verification which is now in progress. After we finish this, the results will be combined with the rest of the biochemical analyses and analyses of oxidative stress and we’ll prepare the manuscript which should be sent for peer review at the beginning of 2012. A part of this study is planned to be presented at the 3rd Feed for health conference in Copenhagen in November 2011.