GcatWiki - User contributions [en]

User:LauraHoopes

2005-12-17T00:22:23Z

LauraHoopes: LH summarizes her involvement with microarrays and qPCR.

Laura Hoopes hopes to discover the elixir of youth before too long, or it will be too late. She encourages any and all communications from GCAT users and friends.

Laura L. Mays Hoopes is the Halstead-Bent Professor of Biology and Molecular Biology at Pomona College in Claremont, CA. She was lucky to get in on the ground floor of GCAT and has used GCAT chips each year of her Genetic Regulation of Eukaryotes class since the laboratory was added in year 2 of the course. In that first year, her class got to be the first undergraduate students to have their data posted on the Stanford Microarray Database, and although the data were not so good, the thrill remained! The summer following that class, LH worked hard on microarrays with Judy Campbell's laboratory group at Caltech, but in year 2 the problems getting good student data persisted. She then got a Wig Grant from the Curriculum Committee to go and visit the important microarray laboratories at Stanford (Barbara Dunn, who was associated with Pat Brown and David Botstein), and at Institute for Systems Biology (Krassen Dimitrov, director of the microarray core facility), contacts suggested by Malcolm Campbell. This trip finally got LH connected with great techniques, and subsequent classes have always had data to analyze, not just gone through the procedure. She also began to work with GCAT to set up a series of workshops so best practices could be discovered and disseminated to faculty using microarrays with undergraduates. In 2004, LH learned to do quantitative reverse transcription PCR on the real time PCR machine, and her research students and technician and she found that the results of many carefully performed microarrays on yeast aging samples, even those with nice looking quality control results in GenePix, were not able to be confirmed by RT PCR. That was not as surprizing as it might have been, since the statistical collaborators on the project Johanna Hardin and her student Alison Wise, had found that using different tests for significant genes had brought out quite different gene lists from the data.
The lab then started using a mRNA amplification strategy that LH had seen used at Stanford and ISB when visiting, and found that results from that method did correspond well with the RT PCR results. The summer, 2005 results using this method gave quite similar gene lists from ANOVA, SAM, and PAM. The course lab tried the mRNA amplification method for the first time in 2005, and used RT PCR for the third year. The 2005 lab was the first whose quantitative PCR results were similar to the microarray results, at least in direction of changes.

GcatWiki:Community Portal

2005-12-17T00:09:18Z

LauraHoopes: Gene Expression in Petite and Malonate-inhibited Yeast

[['''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. 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

File:GCATwikiPetiteMalonate05.doc

2005-12-16T01:19:37Z

LauraHoopes: Genetic Regulation in Eukaryotes at Pomona College analyzed regulation in petite and malonate inhibited cells in 2005.

Genetic Regulation in Eukaryotes at Pomona College analyzed regulation in petite and malonate inhibited cells in 2005.

GcatWiki:Community Portal

2005-12-05T22:56:57Z

LauraHoopes: Petite versus malonate-inhibited yeast gene expression

[['''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