microarray

MicroarraySimultaneously determining the abundance

of multiple(100s-10,000s) transcripts

Gene expression assays

DNA microarrays rely on the hybridization properties of nucleic acids to monitor transcript or RNA abundance on a genome-wide scale in different types of cells.

The main types of gene expression assays:- High-density nylon membrane arrays (macroarrays)- Short oligonucleotide arrays (Affymetrix)- Long oligonucleotide arrays (Agilent)- cDNA arrays- genome arrays

http://www.bioconductor.org/workshops/ShortCourse012302/

http://www.math.neu.edu/~malioutov/sims1.pdf

Uses for Microarray

Determination of transcript (mRNA) abundance

Identification of genes (hypothetical, microRNA)

Diagnostics (disease, stress, chemicals, or pharmaceuticals)

Distinguish signaling pathways

Determination of RNA decay rates


Microarray Experiments; different approaches to compare mRNA

accumulation

Different tissues, same organism (brain versus liver) Same tissue, same organism (treatment versus control, tumor

versus normal) Same tissue, different organisms (wild-type versus knockout or

mutant)

Time course experiments (effect of treatment, development)

Other special designs (e.g. to detect spatial patterns)http://www.bioconductor.org/workshops/ShortCourse012302/

Question

Sample Preparation

Data Analysis

Microarray

Hybridization

Microarray Detection


Overview of the process

Overview of the processMake Chip

Genomic DNA or cDNA

PCR fragment purification and

preparation

Prepare RNA and label cDNA

Grow and/or treat sample

Isolate RNA

cDNA synthesis and labeling

Hybridization

Print array

Data analysis

Genomic versus complementary DNA

Amplification of genomic DNA with

the Polymerase Chain Reaction

(PCR)

Amplification of complementary DNA

(cDNA)with the Polymerase

Chain Reaction(PCR)

http://stein.cshl.org/genome_informatics/guest_lectures_2003/OlgaLecture.pdf

AAAAATTTTTT

mRNA

Oligo-T primerdNTPs

Reverse transcriptase (RT)

RT

DenaturationStep

94oC for 1 min.

Annealing Step45-65oC for 30 sec.

Extension Step 72oC for 2-5 min.

25-30cycles

PCR

Taq

Taq

PCRExponential increase in target DNA

Target DNA

Number of cycles 1 2 3 4

Copies of target DNA2 4 8 16

Building a cDNA chip

Arrayed Library(96 or 384-well plates of bacterial glycerol stocks)

PCR amplification of target DNA

(cDNA or portion of genomic DNA)

Consolidate into plates

Spot as microarrayon glass slides


Make Chip

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Glass slide

http://www.bioconductor.org/workshops/ShortCourse012302/http://www.doc.ic.ac.uk/~yg/course/bioinformatics/2_bioarray-Normalization-lecture.ppt

Microarray Analysis

http://www.genomics.arizona.edu/553/syllabus.html

Label cDNA

Single stranded cDNA (antisense)Cy3 labeled

Reverse transcribe and labelReverse transcriptaseOligo-T primerdNTPs (Cy3 labeled)

Isolate polyA+ RNA from control (wild-type or non-treated)

Single stranded cDNA(antisense)Cy5 labeled

Isolate polyA+ RNA from treatment, mutant, or transgenic organism

Reverse transcribe and labelReverse transcriptaseOligo-T primerdNTPs (Cy5 labeled)

AAAAA AAAAAATTTTTTTTTTTT

Hybridization of nucleic acids

DNA fixed to a matrix matrix = membrane (nylon or nitrocellulose) or glass

5’-GGGCCCATGCATTTAAGCATGGCATGCAT-3’Matrix

III III III III III III II II III III II II II II II II III III II II III III III II II III III II II

3’-CCCGGGTACGTAAATTCGTACCCTACGTA-5’ III III III III III III II II III III II II II II II II III III II II III III III II II III III II II

3’-CCCGGGTACGTAAATTCGTACCCTACGTA-5’

Expression profiling with DNA microarrays

cDNA “control”Cy3 labeled

cDNA “treament”Cy5 labeled

Hybridization Scanning

Laser 1 Laser 2

+

Analysis Image Capture

LABEL

3XSSC

HYB CHAMBER

ARRAY

SLIDE

LIFTERSLIP

SLIDE LABEL

• Humidity• Temperature• Formamide

Hybridization chamber


Microarray Analysis

Data storage and retrieval

Filtering

Normalization

Analysis


Image analysis


Quantification

Cy5 at 635 Cy3 at 532

Overlay images

Expression profiling with DNA microarraysData acquistion

HYBRIDIZATION

Spot #1

Spot #3

Spot #2

Spot #4

Spot #6

Spot #5

Relative intensity

Relative intensity

cDNA “control”Cy3 labeled

6

1

555

5

2

2

2

2 33

Output Output

cDNA “treament”Cy5 labeled

1

6

6

6

5 55

5

3 6

6

4

5

3 6

66

66

6

11

55

5

55

552

2 2

2

33

4

Sample preparation

http://stein.cshl.org/genome_informatics/guest_lectures_2003/OlgaLecture.pdf

Microarray Analysis

Data storage and retrieval

Filtering

Normalization

Analysis

Microarray Analysis

Data Quality

• Irregular size or shape

• Irregular placement• Low intensity

• Saturation• Spot variance• Background

variance

indistinguishable saturated bad print artifactmiss alignment

http://www.mscs.mu.edu/~cstruble/class/mscs230/fall2002/notes/

Replicate spots


Reverse Dye-Label

Reverse-dye label

Reverse-dye label

Microarray AnalysisHierarchical Clustering

http://www.mscs.mu.edu/~cstruble/class/mscs230/fall2002/notes/

Get a Global Perspective;Plot Data on a Metabolic Chart


Data set from an experiment with Arabidopsishttp://www.botany.utoronto.ca/courses/bio472/test_dataset_aracyc.txt

Metabolic pathway analysishttp://www.arabidopsis.org/tools/aracyc/

Microarray Analysis Tutorial Cluster and TreeView


Free download from http://rana.lbl.gov/EisenSoftware.htm

Data set for cluster and treeview http://www.botany.utoronto.ca/courses/bio472/test_dataset.txt

Tutorial as part of a course (outline, test,data and links to free downloads http://www.botany.utoronto.ca/courses/bio472/

Tutorial outlinehttp://www.botany.utoronto.ca/courses/bio472/Microarray_tutorial_WS2004.pdf

microarray

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