MICROARRAY STUDIES ON DICTYOSTELIUM

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MICROARRAY STUDIES ON DICTYOSTELIUM

• Ezgi Okyay
• February 5, 2004

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Summary

• Biology of microarrays
• Basic concepts DNA, RNA, PCR
• Hybridization
• Experimental designs
• Dictyostelium discoideum
• Time course experiments
• Experimenting with the microarrays
• Targets/chips
• Samples

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DNA - RNA

• http//www.kubrussel.ac.be/onderwijs/etew/tew/vakk
  en/tweedekan/ecsector/biotech/

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RT-PCR
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What are we working with?

• Dictyostelium discoideium
• Eukaryotic model organism
• Unicellular while growing (vegitative)
• Multicellular during development
• 24 hr development

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Development
8h
12h
16h
24h
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Experimental Design I(sample)

• While cells are developing
• Collect cells every 2 hrs 0,2,4,,24hrs
• Isolate RNA from each sample
• Label them with fluorescent dye
• Mix with reference labeled with fluorescent dye
• RT-PCR into DNA

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Experimental Design II(target microarray)

• Target sequence preparation
• Target slide preparation
• Target printing
• Hybridization of sample/chip

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Sleeping stocks at 80 C
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Cells waking up
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Cell growth
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(No Transcript)
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Starving cells - Development

• 0.5 ml of cell culture (108 cells)
• Develop on filters

8h
12h
16h
24h
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RNA isolation from cells

• Cells are grown to a concentration of 108
  cells/ml (yield 100-1000ug of RNA)
• Spun down in centrifuge
• Washed
• Resuspended in TRIZOL
• RNA extraction using
• Phenol/chloroform method
• RNA washed,put in buffer
• RNAse free

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Reverse Transcription Steps

• 10 ug RNA mixed with 0.5 ug primer incubated at
  70 C for 10 min.
• Incubated on ice for 3-5min.
• Incubated at 42 C for 2 hrs after adding reaction
  buffer (Fl-dNTP,dNTP,Superscript II )
• Reaction terminated by addition of EDTA
• RNA degraded by adding NaOH and incubating at 60
  C for 20 min.
• DNA precipitated by adding sodium acetate and
  ethanol

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The samples

• DNA from each 13 time point samples
• Label them with Cy5 red fluorescent label
• Labeled reference with Cy3 green fluorescent
  label
• Reference contain mixed DNA from 0,3,6,12,17 and
  24 hrs
• Cy3/Cy5 mixed in one tube per time point

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What do we have?

• 13 time point samples are collected
• 13 time point samples are labeled
• Ready to hybridize with microarray
• Need to have microarrays
• Targets printed on them

What do we need?
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Microarray preparations

• Glass slides
• Washed in acetone with sonication for 10 min
• Rinsed with distilled water
• Immersed in glycidoxypropyltrimethoxysile

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Microarray preparations

• Washed in 100 Ethanol
• Dried with Nitrogen
• Baked at 100 C for gt 2hrs
• Ready for printing

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Target preparation

• Grow clones
• PCR
• Check size
• Isolate DNA
• Sequence
• Prepare plates

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Target preparation

• Check size
• Isolate DNA

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Target preparation

• Sequence
• Prepare plates

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Printing Targets on Slides
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Print Layout
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Hybridization
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After hybridization
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Imaging

• Slides scanned for both channels separately
• Outputting two files for cy3/cy5
• Analysis made using these two files
• Log(cy3/cy5) ratios are used for analysis