BIOL398-01/S10:DNA Microarrays

Background

References

1. Brown PO and Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet. 1999 Jan;21(1 Suppl):33-7. DOI:10.1038/4462 |

   Brown & Botstein (1999) link to full text

2. Salomonis N, Hanspers K, Zambon AC, Vranizan K, Lawlor SC, Dahlquist KD, Doniger SW, Stuart J, Conklin BR, and Pico AR. GenMAPP 2: new features and resources for pathway analysis. BMC Bioinformatics. 2007 Jun 24;8:217. DOI:10.1186/1471-2105-8-217 |

   Salomonis et al. (2007) link to full text

3. Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC, and Conklin BR. GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nat Genet. 2002 May;31(1):19-20. DOI:10.1038/ng0502-19 |

   Dahlquist et al. (2002) link to full text

4. Doniger SW, Salomonis N, Dahlquist KD, Vranizan K, Lawlor SC, and Conklin BR. MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data. Genome Biol. 2003;4(1):R7. DOI:10.1186/gb-2003-4-1-r7 |

   Doniger et al. (2003) link to full text

5. LaTulippe E, Satagopan J, Smith A, Scher H, Scardino P, Reuter V, and Gerald WL. Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease. Cancer Res. 2002 Aug 1;62(15):4499-506.

   LaTulippe et al. (2002) link to full text, dataset (on this wiki)

6. Merrell DS, Butler SM, Qadri F, Dolganov NA, Alam A, Cohen MB, Calderwood SB, Schoolnik GK, and Camilli A. Host-induced epidemic spread of the cholera bacterium. Nature. 2002 Jun 6;417(6889):642-5. DOI:10.1038/nature00778 |

   Merrell et al. (2002) link to full text, dataset (on this wiki)

7. van de Mortel JE, Almar Villanueva L, Schat H, Kwekkeboom J, Coughlan S, Moerland PD, Ver Loren van Themaat E, Koornneef M, and Aarts MG. Large expression differences in genes for iron and zinc homeostasis, stress response, and lignin biosynthesis distinguish roots of Arabidopsis thaliana and the related metal hyperaccumulator Thlaspi caerulescens. Plant Physiol. 2006 Nov;142(3):1127-47. DOI:10.1104/pp.106.082073 |

   van de Mortel et al. (2006) link to full text, dataset (on this wiki)

8. O'Neill AJ, Lindsay JA, Gould K, Hinds J, and Chopra I. Transcriptional signature following inhibition of early-stage cell wall biosynthesis in Staphylococcus aureus. Antimicrob Agents Chemother. 2009 Apr;53(4):1701-4. DOI:10.1128/AAC.01309-08 |

   O'Neill et al. (2009) link to full text, dataset (on this wiki)

9. Tai SL, Daran-Lapujade P, Walsh MC, Pronk JT, and Daran JM. Acclimation of Saccharomyces cerevisiae to low temperature: a chemostat-based transcriptome analysis. Mol Biol Cell. 2007 Dec;18(12):5100-12. DOI:10.1091/mbc.e07-02-0131 |

   Tai et al. (2007) link to full text, dataset (on this wiki)

Groups

• Michael, Kris, Salomon: Arabidopsis thaliana, van de Mortel et al. (2006)
• Janelle, KP: human prostate cancer, LaTulippe et al. (2002)
• J'aime, Amanda: Vibrio cholerae, Merrell et al. (2002)
• Alex, Bobak: Saccharomyces cerevisiae, Tai et al. (2007)
• Angela, Ryan: Staphylococcus aureus, O'Neill et al. (2009)

Week 11

• Slides shown in class on 4/6/10 are available on MyLMUConnect under "Content".

Overview of Microarray Data Analysis

This is a list of steps required to analyze DNA microarray data.

1. Quantitate the fluorescence signal in each spot in the microarray image.
   • Typically performed by the scanner software, although third party software packages do exist.
   • The image of the microarray slide and this quantitation are considered the "raw-est" form of the data.
   • Ideally, this type of raw data would be made publicly available upon publication.
   • In practice, the image data is usually not made available because the raw image file of one slide could be up to 100 MB in size.
   • Also, some journals do not require data deposition as a requirement for publication, so often published data are not actually available anywhere for download.
   • Microarray data is not centrally located on the web. Some major sources are:
     • NCBI GEO
     • EBI ArrayExpress
     • Stanford Microarray Database
     • PUMAdb (Princeton Microarray Database)
     • In addition, microarray data can sometimes be found as supplementary information with a journal article or on an investigator's own web site.
2. Calculate the ratio of red/green fluorescence
3. Log(base 2) transform the ratios
4. Normalize the log ratios on each microarray slide
5. Normalize the log ratios for a set of slides in an experiment
6. Perform statistical analysis on the log ratios
7. Compare individual genes with known data
8. Look for patterns (expression profiles) in the data (many programs are available to do this)
9. Perform Gene Ontology term enrichment analysis (we will use MAPPFinder for this)
10. Map onto biological pathways (we will use GenMAPP for this)

Preparation for Next Week's Journal Club

In preparation for the Journal Club, each individual will do the following assignment on their individual Week 11 Journal page.

1. Make a list of at least 10 biological terms for which you did not know the definitions when you first read the article. Define each of the terms. You can use the glossary in any molecular biology, cell biology, or genetics text book as a source for definitions, or you can use one of many available online biological dictionaries (links below). List the citation(s) for the dictionary(s) you use, providing a URL to the page is fine.
2. Write an outline of the article. The length should be the equivalent of 2 pages of standard 8 1/2 by 11 inch paper. Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article.
   • What is the main result presented in this paper? (Hint: look at the last sentence of the introduction and restate it in plain English.)
   • What is the importance or significance of this work?
   • What were the limitations in previous studies that led them to perform this work?
   • What were the methods used in the study?
     • What samples did they collect and use for the microarray experiment?
     • How many microarray chips did they hybridize in the experiment?
     • Which samples were paired to hybridize on the chip?
     • Which was labeled red (Cy5)? Which was labeled green (Cy3)?
     • How many replicates did they perform of each type?
       • Biological replicates are made from entirely different biological samples.
       • Technical replicates are made when one biological sample is split at a particular stage in the procedure and then carried through to the end of the procedure.
     • What do they say about how they performed each of the steps listed in the Overview of Microarray Data Analysis section above?
   • Briefly state the result shown in each of the figures and tables.
   • How do the results of this study compare to the results of previous studies (See Discussion).
3. Upload your completed PowerPoint slides to your journal page by the Week 11 journal deadline (you may make changes before your presentation Tuesday morning, but I will be evaluating the presenttion you upload.)

Get Acquainted with Your Microarray Dataset

• Find the web site or database where your paper's microarray data are available.
• Download your dataset from this wiki and open it in Excel.
• Match the columns of data with your description of the experimental design from your outline.

Week 12

• Journal Club presentations on your group's paper.
• Statistical analysis of the microarray data
  • Sample Microarray Analysis based on Merrell et al. (2002) Vibrio cholerae data

Week 13

• Project work session
  • GenMAPP and MAPPFinder Protocols
  • For the groups working with Arabidopsis thaliana, Staphylococcus aureus, and Vibrio cholerae, you will need to download the GenMAPP Gene Database for those species from the XMLPipeDB SourceForge site.

Week 14

• Final project presentations in class.
• Course evaluations