Transcript Todd Eckdahl - Davidson College

Using DNA Microarrays from
Multiple Species:
Comparisons for Teaching
Effectiveness
Todd T. Eckdahl
Biology Department
Missouri Western State College
Overview






Background
Courses using microarray technology
Species studied
Implementation
Results
Planned research projects
Missouri Western State College
 Saint Joseph, Missouri
 State-supported PUI
 ~5200 students
 200 faculty
 Biology Department




~340 majors
10 faculty
No graduate degree programs
New major in Biochemistry and Molecular Biology
Courses Using Microarray
Technology
 BIO 431 Molecular Biology
 4 credit course
 3 hours lecture, 3 hours lab
 Student majors
 BMB, Biology with Health Sciences emphasis
 BIO 313 Topics in Molecular Genetics
 1 credit course
 3 hours lab
 Student majors
 BMB, Biology-Health Sciences, Teaching
Functional Genomics
Technology
 Microarrays
 cDNAs printed
 eg. Stanford yeast chips, UW E. coli chips
 80-mer oligos printed
 eg. ISB yeast chips
 Labeling options
 Indirect labeling
 eg. Genisphere dendrimers
 Direct incorporation
 eg. Ulysis alexafluore labeling
Conducting Microarray
Experiments in a Course
 Emphasize the “Big Picture”
 Genomics, functional genomics, proteomics
 Shift to data-rich science





Primary Literature
Brainstorming for ideas
Scheduling
Data Analysis
Presentations
Ideas for Yeast
Experiments
 Glucose vs. Galactose vs. Fructose
vs. Maltose
 Anaerobic vs. aerobic
 Induction of sporulation
 Heat Shock v. Cold Shock
 Drug treatment
Minor Groove Binding Drugs
 Anti-tumor properties
 Conformational change in the 3D structure
of DNA
 Prior Knowledge of MGBD/DNA
interaction
 As models for minor
groove binding proteins
DAPI
Yeast Culture




OD at 660 nm to measure turbidity
Grown through log phase
4 hours of exposure to 10 uM DAPI
Control culture without DAPI
Isolation of RNA
 Sterile, RNase- free equipment and
workspace
 Harvesting of yeast
 Production of spheroblasts
 Isolation of RNA via RNA spin
column
 Elution of RNA
 Quantify RNA with A260 / A280
 Run RNA on denaturing agarose
Preparation of labeled
cDNA and hybridization
 Reverse transcription of RNA
 capture sequence incorporated
 Label preparation
 addition of Cy3 and Cy5 dendrimer
 addition of capturing reagents
 Add probe to slide, cover and
incubate at 55 C for 1-3 days
Experimental Summary
Yeast in log phase
untreated
10 uM DAPI
Total RNA
Total RNA
Reverse Txn
cDNA
cDNA
Red
fluorophore
Green
fluorophore
microarray
hybridization
Data
Acquisition
 Post-hybe wash,
dry slide
 Ship for
scanning
 Receive data
 Scanalyze
 Submit to SMD
Microarray Controls






Empty or 3X SSC
Duplicate genes
Duplicate experiments
Cy3 and Cy5 dyes
Poly A
Genomic, Intron, tRNA
Example of induced gene
YBR012W-B, TyB Gag-Pol protein
TGAGAAGCTGTCATCGAAGTTAGAGGAAGCTGAAGTGCAAGGATTGATAA
TGTAATAGGATAATGAAACATATAAAACGGAATGAGGAATAATCGCAATA
TTAGTATGTAGAAATATAGATTCCATTTTGAGGATTCCTATATCCTCGAG
GAGAACTTCTAGTATATTCTGTATACCTAATATTATAGCCTTTATCAACA
ATG
Example of repressed
gene
YHR055C, copper-binding metallothionein
TTCCGCTGAACCGTTCCAGCAAAAAAGACTACCAACGCAATATGGATTGT
CAGAATCATATAAAAGAGAAGCAAATAACTCCTTGTCTTGTATCAATTGC
ATTATAATATCTTCTTGTTAGTGCAATATCATATAGAAGTCATCGAAATA
GATATTAAGAAAAACAAACTGTACAATCAATCAATCAATCATCACATAAA
ATG
Analyses at Stanford
Microarray Database
 Single spot or sequence
 Data filtering
 signal strength
 R/G or G/R ratio
 Linear regression comparison
 Prepare data for clustering
Databases linked to SMD
 SGD - Saccharomyces genome
database
 Genbank
 YPD - yeast protein database
 Swissprot protein database
Ideas for E. coli Experiments
 Metabolic shift
 Osmotic stress
 Growth curve effects
 Heat Shock v. Cold Shock
 Drug treatment
 Effects of gene deletion
BIO 313 Experiment
 E coli chips
 M1655 sequenced strain
 cDNA spotted
 Putative transcriptional regulators
 nusA deletion strain
 yhbM deletion strain
 Two channel hybridizations
 Compare labeled RNA from wt versus deletion
E. coli culture
 Overnight culture
 Grown at 37°C to log phase
 OD at 600 nm to measure turbidity
RNA Isolation
 Sterile, RNase-free equipment and
work area
 Total RNA SafeKit
 Total RNA Safe protocol used
 Lysis of E. coli done with mixture of
TE and lysozyme
RNA Isolates
 Measure A260 / A280
 Check on denaturing
agarose gel
Labeling
 Labeling of isolated RNA done by use
of ULYSIS Nucleic Acid Direct
Labeling Kit
 ULYSIS protocol followed
 Fluorescent Dyes: Alexa Fluor 546
(green) and Alexa Fluor 660 (red)
Hybridization
 Microarray prehybridized
 Labeled RNA mixed together in
hybridization buffer and added to
slide
 Hyb at 55 C in dry incubator
overnight
 Post-hyb washes
Microarray Controls





Empty or 3X SSC
Duplicate genes
Duplicate experiments
Cy3 and Cy5 dyes
Genomic
Examples of Results
asr
flhC
emrY
Examples
 Induced
 Asr, G1787881
 acid shock protein
 Repressed
 flhC, G1788201
 regulator of flagellar biosynthesis acting on class 2
operons
 Non-responsive
 emrY, G1788710
 multidrug resistance protein Y
 putative transport
Example of induced gene
Asr, G1787881
gatca agactactattattggtagctaaatttcccttaagtcac
aatacgttattatcaacgctgtaatttattcagcgtttg
tacatatcgttacacgctgaaaccaaccactcacggaag
tctgccattcccagggatatagttatttcaacggccccg
cagtggggttaaatgaaaaaacaaattgagggtatgaca
1 - atg aaa aaa gta tta gct ctg gtt gtt gcc
31 - gct gct atg ggt ctg tct tct gcc gcc ttt
61 - gct gca gag act acg acc aca cct gct ccg
91 - act gcg acg acc acc aaa gca gcg ccg gcg
Example of repressed
gene
flhC, G1788201
ccgca aatggttaagctggcagaaaccaatcaactggtttgtca
cttccgttttgacagccaccagacgattactcagttgac
gcaagattcccgcgttgacgatctccagcaaattcatac
cggcatcatgctctcaacacgcttgctgaatgatgttaa
tcagcctgaagaagcgctgcgcaagaaaagggcctgatc
1 - atg agt gaa aaa agc att gtt cag gaa gcg
31 - cgg gat att cag ctg gca atg gaa ttg atc
61 - acc ctg ggc gct cgt ttg cag atg ctg gaa
91 - agc gaa aca cag tta agt cgc gga cgc ctg
Example of non-responsive
gene
emrY, G1788710
gaact catggaacaccccttgcgtattggtttatcgatgacagc
aactattgatacgaagaacgaagacattgccgagatgcc
tgagctggcttcaaccgtgacctccatgccggcttatac
cagtaaggctttagttatcgataccagtccgatagaaaa
agaaattagcaacattatttcgcataatggacaacttta
1 - atg gca atc act aaa tca act ccg gca cca
31 - tta acc ggt ggg acg tta tgg tgc gtc act
61 - att gca ttg tca tta gcg aca ttt atg caa
91 - atg ttg gat tcc act att tct aac gtc gca
Microarrays in Courses:
Lessons Learned
 Advance planning essential
 Controls for critical steps
 Reliability and Reproducibility
 Do Controls Make Sense?
 Do Results Make Sense?
 Potential for large amounts of data
means extensive analysis time
needed
Ongoing and Planned
Research Projects
 Measure Effects of
Minor Groove
Binding Drugs on
Gene Expression in
Yeast
 Measure Effects of
Minor Groove
Binding Drugs on
Gene Expression in
Human Tumor Cells
in Culture
Big Ideas
 Sequence and structural
requirements for MGBD binding
 A+T rich sequences
 DNA bending
 Determination of optimal binding sites
 Effects of MGBDs on gene expression
 Preliminary data using RT-PCR
 Global patterns of gene expression
 Complementary in vitro and in vivo
approaches
Acknowledgements
 Genome Consortium for Active
Teaching
 Malcolm Campbell, Davidson College
 NSF DBI 0099720 MUE grant
 Dr. Barbara Dunn, Stanford
University
 Dr. Fred Blattner lab, UW-Madison
 Dr. Bob Getts, Genisphere, Inc.
 Missouri Western Students