1. Overview of Microarray

2. 2/71 Gene Expression Gene expression Production of mRNA is very much a reflection of the activity level of gene In the past, looking at whether a specific gene is turned up (upregulated) or turned off (downregulated) under certain condition

3. 3/71 Microarray Data Microarray: a technological advancement study the genes of an organism’s at once Microarrays are a massively-parallel Northern Blot High throughput method allow for the global study of changes in gene expression → complete cellular snapshot

4. 4/71 Reverse Transcription Clone cDNA strands, complementary to the mRNA

5. 5/71 Microarray Experiments mRNA levels compared in many different contexts Different tissues, same organism (brain vs. liver) Same tissue, same organism (tumor vs. non-tumor) Same tissue, different organisms (wild-type or mutant) Time course experiments (development)

6. 6/71 Expression Profiles in Tissues In any type of tissue, only a limited set of the genes are switched on at any given time. Also, from one tissue type to another, the limited set of genes involved will vary. Thus, each tissue can be identified by its unique pattern of gene expression. This pattern is often called an “expression profile” or a “molecular signature”. Here is an example of a normal breast cell and a normal prostate cell. Although both of these cells have many mRNAs and proteins in common (grey), they also have unique differences.

7. 7/71 Expression Profiles in Cancer It is possible to measure differences between a normal and a cancer tissue of the same type--- for example, normal and cancerous prostate. When a normal prostate tissue is transformed into cancerous prostate tissue, the expression profile changes.  Any changes in gene expression ultimately cause alterations in protein production.  New expression profiles in a cancer cell can dramatically alter the network of proteins that interact.  A critical protein may no longer be available, another may be overproduced, yet another may be flawed. And when new genes become activated, entirely new proteins may be introduced. Many different combinations of gene changes and protein interactions are seen in cancerous tissue.

8. 8/71 Cells and Gene Expression The repertoire of gene products produced by a cancer cell might differ in two ways from its normal counterpart: Quantitatively As shown for gene B, which is expressed at an abnormally high level, and gene A, which is not expressed at all. Qualitatively As shown for gene C*, which is mutated such that it produces an altered gene product.

9. 9/71 Two Main Technologies for Making Microarrays Robotic spotting From D. Steke ’ s Microarray Bioinformatics

10. 10/71 nylon array 10pmol/mm 2 glass array 0.1pmol/mm 2 From prof. 陳同孝 ’ s slide

11. 11/71 Two Main Technologies for Making Microarrays (cont’d) In situ synthesis Using photolithography http://www.affymetrix.com/technology/manufacturing/index.affx

12. 12/71 Two Type of Microarrays (Harrington et al. 2000) cDNA Array Oligo. Array

13. 13/71 cDNA Array (Harrington et al. 2000)

14. 14/71 cDNA Probe Preparation http://www.accessexcellence.org/AB/GG/polymerase.html

15. 15/71 Sample Preparation Compare the genetic expression in two samples of cells SAMPLES cDNA labelled red/green

16. 16/71 Dye Labelling aminoally-dUTP

17. 17/71 Hybridization and Scanning HYBRIDIZE Add equal amounts of labelled cDNA samples to microarray. SCAN Expression profiling using cDNA microarrays Nature, 21, 1999

18. 18/71 http://www.iridian.ca/products/products_fluorescence.htm

19. 19/71 cDNA Array (cont’d) cDNA Array: e.g. Agilent One probe  one gene Processing steps: Experimental design Sample preparation RNA extraction Prepare cDNA Labeling cDNA with dye Hybridization Quantitation Hybridization Microarray manufacturing Sample preparation Quantitation Data analysis Experiment Probe preparation

20. 20/71 cDNA Array (cont’d) http://www.bioteach.ubc.ca/MolecularBiology/microarray/

21. 21/71 Oligonucleotide Array Synthesized on a chip: e.g. Affymetrix Using photolithography Each gene may have several probe sets; each probe sets have above 10 probes. http://www.affymetrix.com/technology/manufacturing/index.affx

22. 22/71 Cross-Hybridization

23. 23/71 PM/MM

24. 24/71 PM/MM (cont’d)

25. 25/71 Oligonucleotide Array (cont’d) http://www.bioteach.ubc.ca/MolecularBiology/microarray/

26. 26/71 Oligonucleotide Array (cont’d)

27. 27/71 Oligonucleotide Array (cont’d) http://www.bioteach.ubc.ca/MolecularBiology/microarray/

28. 28/71 cDNA Array vs. Oligo. Array Probes are cDNA fragments, usually amplified by PCR. At least two samples are hybridized to chip. One probe one gene. Probes of varying length Fluorescence at different wavelengths measured by a scanner. Probes are deposited on a solid support, either positively charged nylon or glass slide. Probes are oligos synthesized in situ using a photolithographic approach. One target sample per array. 16-20 probe-pairs per gene. Probes are 25-mers. The apparatus requires a fluidics station for hybridization and a special scanner. There are at least 5 oligos per cDNA, plus an equal number of negative controls. From Dr. 吳漢銘 ’ s slide

29. 29/71 Advantages and Disadvantages of cDNA Array compared with Oligo. Array Advantages Can choose the DNA on the array Cheaper Fluorescence at different wavelengths measured by a scanner Can hybridize closely related species Disadvantages Less specificity (will cross hybridize to genes ~80% homology) Cannot distinguish closely related gene families May need to confirm DNA sequence Repeated amplification and quality control From Dr. 吳漢銘 ’ s slide

30. 30/71 Advantages and Disadvantages of cDNA Array compared with Oligo. Array Advantages High specificity (small probe length means gene family members can be differentiated) Very robust protocols and results are very reproducible Can use small amount of RNA Widely used, so annotation of probe sets is of relatively high quality Disadvantages Very expensive to design (~US$300,000) Expensive to perform experiments (~US$400 + $300 labeling/hybridization) Limited to the species for which there are chips available sequence required Single target hybridization, so comparison always involves two experiments, and dye swaps are impossible Match/mismatch technology has major limitations: mismatch signal often higher than match, and dose response curve is different for each pair From Dr. 吳漢銘 ’ s slide

31. 31/71 Microarray Experimental Flowchart R=Rf-Rb G=Gf-Gb M=log2R/G A=1/2 log2RG Microarray Life Cycle