LauraHoopes: Gene Expression in Petite and Malonate-inhibited Yeast

2005-12-17T00:09:18Z

Gene Expression in Petite and Malonate-inhibited Yeast

LauraHoopes: Petite versus malonate-inhibited yeast gene expression

2005-12-05T22:56:57Z

Petite versus malonate-inhibited yeast gene expression

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[['''GENETIC REGULATION IN EUKARYOTES, POMONA COLLEGE, 2005 PROJECT: Petite and malonate-inhibited yeast gene expression.''']]
This project is an investigation of the gene expression patterns in wild type yeast, wild type yeast inhibited with malonate (an inhibitor of TCA cycle Succinate Dehydrogenase enzymes) and yeast selected to be petite. Petites have lost some or all of the mitochondrial genome and have major changes in gene expression under some culture conditions(Epstein et al, Mol Biol of the Cell 12:297-305, 2001). The class experiment explored gene expression in these three conditions using glucose as the carbon source, unlike Epstein et al., and we also hoped to study petites treated with malonate, for which little data were obtained due to a failure of amplification of RNA for the microarrays. For each condition, the class performed RNA isolation and quality control, used an Ambion kit to amplify the mRNA and couple the amplified RNA to Cy3 or Cy5, hybridized duplicate but dye-flipped GCAT arrays printed at WU, and analyzed the data using GenePix for quality control and GeneSpring for clustering and ANOVA. Once we learn how, we will be posting our data and analysis for other GCAT users to see and comment upon. We also ran RT PCR of two genes the class picked from the array data to see if we could verify our results. Results from that will also be posted later, after we learn how.
[[Image:Cell pellets from Wild Type, Wild Type Malonate, Petite, and Petite Malonate

]]
Laura Hoopes

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    [['''GENETIC REGULATION IN EUKARYOTES, POMONA COLLEGE, 2005 PROJECT: Petite and malonate-inhibited yeast gene expression.''']]
-This project is an investigation of the gene expression patterns in wild type yeast, wild type yeast inhibited with malonate (an inhibitor of TCA cycle Succinate Dehydrogenase enzymes) and yeast selected to be petite.  Petites have lost some or all of the mitochondrial genome and have major changes in gene expression under some culture conditions(Epstein et al, Mol Biol of the Cell 12:297-305, 2001).  The class experiment explored gene expression in these three conditions using glucose as the carbon source, unlike Epstein et al., and we also hoped to study petites treated with malonate, for which little data were obtained due to a failure of amplification of RNA for the microarrays.  For each condition, the class performed RNA isolation and quality control, used an Ambion kit to amplify the mRNA and couple the amplified RNA to Cy3 or Cy5, hybridized duplicate but dye-flipped GCAT arrays printed at WU, and analyzed the data using GenePix for quality control and GeneSpring for clustering and ANOVA.  Once we learn how, we will be posting our data and analysis for other GCAT users to see and comment upon. We also ran RT PCR of two genes the class picked from the array data to see if we could verify our results. Results from that will also be posted later, after we learn how.
+This project is an investigation of the gene expression patterns in wild type yeast, wild type yeast inhibited with malonate (an inhibitor of TCA cycle Succinate Dehydrogenase enzymes) and yeast selected to be petite.  Petites have lost some or all of the mitochondrial genome and have major changes in gene expression under some culture conditions(Epstein et al, Mol Biol of the Cell 12:297-305, 2001).  The class experiment explored gene expression in these three conditions using glucose as the carbon source, unlike Epstein et al., and we also hoped to study petites treated with malonate, for which little data were obtained due to a failure of amplification of RNA for the microarrays.  For each condition, the class performed RNA isolation and quality control, used an Ambion kit to amplify the mRNA and couple the amplified RNA to Cy3 or Cy5, hybridized duplicate but dye-flipped GCAT arrays printed at WU, and analyzed the data using GenePix for quality control and GeneSpring for clustering and ANOVA.  We also ran RT PCR of two genes the class picked from the array data to see if we could verify our results.  Results and further description can be found on the class web page by choosing the Petite Malconate Project. [http://pages.pomona.edu/~llh04747/genreg.html]
 Laura Hoopes

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LauraHoopes: Gene Expression in Petite and Malonate-inhibited Yeast

LauraHoopes: Petite versus malonate-inhibited yeast gene expression