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Microarray Simultaneously determining the abundance of multiple(100s-10,000s) transcripts.

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Presentation on theme: "Microarray Simultaneously determining the abundance of multiple(100s-10,000s) transcripts."— Presentation transcript:

1 Microarray Simultaneously determining the abundance of multiple(100s-10,000s) transcripts

2 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/

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

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

5 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/

6 Question Sample Preparation Data Analysis Microarray Hybridization Microarray Detection http://www.bioconductor.org/workshops/ShortCourse012302/ Overview of the process

7 Make 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

8 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 AAAAA TTTTTT mRNA Oligo-T primer dNTPs Reverse transcriptase (RT) RT

9 Denaturation Step 94 o C for 1 min. Annealing Step 45-65 o C for 30 sec. Extension Step 72 o C for 2-5 min. 25-30 cycles PCR Taq

10 PCR Exponential increase in target DNA Target DNA Number of cycles 12 3 4 Copies of target DNA 2 4 8 16

11 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 microarray on glass slides http://www.bioconductor.org/workshops/ShortCourse012302/

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

13 Microarray Analysis http://www.genomics.arizona.edu/553/syllabus.html

14 Label cDNA Single stranded cDNA (antisense) Cy3 labeled Reverse transcribe and label Reverse transcriptase Oligo-T primer dNTPs (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 label Reverse transcriptase Oligo-T primer dNTPs (Cy5 labeled) AAAAAAAAAAA TTTTTT

15 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’

16 Expression profiling with DNA microarrays cDNA “control” Cy3 labeled cDNA “treament” Cy5 labeled Hybridization Scanning Laser 1 Laser 2 + AnalysisImage Capture

17 LABEL 3XSSC HYB CHAMBER ARRAY SLIDE LIFTERSLIP SLIDE LABEL Humidity Temperature Formamide Hybridization chamber http://www.bioconductor.org/workshops/ShortCourse012302/

18 Microarray Analysis Data storage and retrieval Filtering Normalization Analysis http://www.bioconductor.org/workshops/ShortCourse012302/

19 Image analysis http://www.bioconductor.org/workshops/ShortCourse012302/

20 Quantification Cy5 at 635Cy3 at 532 Overlay images

21 Expression profiling with DNA microarrays Data acquistion HYBRIDIZATION Spot #1 Spot #3 Spot #2 Spot #4 Spot #6 Spot #5 Relative intensity Relative intensity cDNA “control” Cy3 labeled 6 1 5 5 5 5 2 2 2 2 3 3 Output cDNA “treament” Cy5 labeled 1 6 6 6 5 5 5 5 3 6 6 4 5 3 6 6 6 6 6 6 1 1 5 5 5 5 5 5 5 2 2 2 2 3 3 4

22 Sample preparation http://stein.cshl.org/genome_informatics/guest_lectures_2003/OlgaLecture.pdf

23 Microarray Analysis Data storage and retrieval Filtering Normalization Analysis

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

25

26

27

28 Microarray Analysis Data Quality Irregular size or shape Irregular placement Low intensity Saturation Spot variance Background variance indistinguishablesaturated bad print artifactmiss alignment http://www.mscs.mu.edu/~cstruble/class/mscs230/fall2002/notes/

29 Replicate spots http://www.genomics.arizona.edu/553/syllabus.html

30 Reverse Dye-Label Reverse-dye label

31 Microarray Analysis Hierarchical Clustering http://www.mscs.mu.edu/~cstruble/class/mscs230/fall2002/notes/

32 Get a Global Perspective; Plot Data on a Metabolic Chart http://www.genomics.arizona.edu/553/syllabus.html Data set from an experiment with Arabidopsis http://www.botany.utoronto.ca/courses/bio472/test_ dataset_aracyc.txt Metabolic pathway analysis http://www.arabidopsis.org/tools/aracyc/

33 Microarray Analysis Tutorial Cluster and TreeView http://www.genomics.arizona.edu/553/syllabus.html Free download from http://rana.lbl.gov/EisenSoftware.htm Data set for cluster and treeview http://www.botany.utoronto.ca/courses/bio472/test_dat aset.txt Tutorial as part of a course (outline, test,data and links to free downloads http://www.botany.utoronto.ca/courses/bio472/ Tutorial outline http://www.botany.utoronto.ca/courses/bio472/Microarr ay_tutorial_WS2004.pdf

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