Microarray

microarray technology

1. Microarray Dr. Swarnendu Pal

2. Background History Introduction Types of microarrays DNA microarray Principle Types of DNA microarray Steps involved in DNA microarray Applications Advantages Limitations and disadvantages Future of microarrays Take home message

3. Background A human organism has over 250 different cell types (e.g., muscle, skin, bone , neuron ), most of which have identical genomes, yet they look different and have different activities Less than 20% of the genes are expressed in a typical cell type Apparently the differences in gene expression is what makes the cells look and function differently

4. History Once the human genome sequence was completed in 2001, it paved the way for many experiments and researches; one such area was identifying the regions of DNA which control normal and disease states. Functional genomics is the study of gene function through parallel expression measurements of a genome.

5. History Microarray  analyzes large amount of samples which have either been recorded previously or from new samples History: Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known DNA sequence Use of miniaturized microarrays for gene expression profiling first reported in 1995, and a complete eukaryotic genome ( Saccharomyces cerevisiae ) on a microarray was published in 1997

6. Microarray It is a 2D array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high-throughput screening, miniaturized, multiplexed and parallel processing and detection methods.

7. Types DNA microarrays , such as cDNA microarrays, oligonucleotide microarrays, BAC microarrays and SNP microarrays MMChips , for surveillance of microRNA populations Protein microarrays Peptide microarrays , for detailed analyses or optimization of protein–protein interactions Tissue microarrays Cellular microarrays (also called transfection microarrays) Chemical compound microarrays Antibody microarrays

8. DNA microarray A DNA microarray (also commonly known as genome chip, DNA chip, or gene array ) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. Each spot of DNA, called a probe , represents a single gene.

9. Principle Hybridization : The property of complementary nucleic acid sequences is to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization.

10. Types of DNA microarrays Based on different technologies and probe types, and intended application it can be: Bacterial artificial chromosome arrays cDNA microarrays Oligonucleotide microarrays In-situ synthesized microarrays Bead microarrays

11. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data

12. Instruments required

13. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Collecting tissue

14. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating RNA

15. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating RNA

16. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating RNA

17. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating RNA

18. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating RNA

19. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating mRNA

20. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating mRNA

21. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating mRNA

22. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Isolating mRNA

23. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Make labeled DNA copy

24. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Make labeled DNA copy

25. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Make labeled DNA copy

26. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Make labeled DNA copy

27. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Apply DNA

28. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Apply DNA

29. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Apply DNA

30. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Apply DNA

31. Steps involved In microarray Collect tissue Isolate RNA Isolate mRNA Make labeled DNA copy Apply DNA Scan microarray Analyze data Scan microarray

32. Analyzing data GREEN represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. RED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA. YELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA. BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA.

33. Analyzing data

34. Image Analysis & Data Visualization

35. Some questions for the age of genomics How gene expression differs in different cell types? How gene expression differs in a normal and diseased (e.g., cancerous) cell? How gene expression changes when a cell is treated by a drug? How gene expression changes when the organism develops and cells are differentiating?

36. Applications In cancer Tumor formation involves simultaneous changes in hundreds of cells and variations in genes. Identification of single-nucleotide polymorphisms (SNPs) and mutations, classification of tumors , identification of target genes of tumor suppressors,

37. Applications In cancer Identification of cancer biomarkers, identification of genes associated with chemoresistance Early detection of precancerous lesions Identification of gene expression profiles or “genomic fingerprints” will allow clinicians to differentiate harmless lesions from precancerous lesions or from very early cancer

38. Applications Antibiotic treatment Gene expression profiling In different cells/tissues During the course of development Under different environmental or chemical stimuli In disease state versus healthy Molecular diagnosis Molecular classification of disease Drug development Identification of new targets Pharmacogenomics Individualized medicine

39. Advantages Provides data for thousands of genes. One experiment instead of many. Fast and easy to obtain results. Huge step closer to discovering cures for diseases and cancer. Different parts of DNA can be used to study gene expresion

40. Limitations of DNA microarrays First, arrays provide an indirect measure of relative concentration. However, due to the kinetics of hybridization, the signal level at a given location on the array is not linearly proportional to concentration of the species hybridizing to the array. For complex mammalian genomes, it is often difficult to design arrays in which multiple related DNA/RNA sequences will not bind to the same probe on the array.

41. Disadvantages The biggest disadvantage of DNA chips is that they are expensive to create. The production of too many results at a time requires long time for analysis, which is quite complex in nature. The DNA chips do not have very long shelf life, which proves to be another major disadvantage of the technology

42. The Future of DNA arrays When the cost is similar, sequencing has many advantages relative to microarrays. Sequencing is a direct measurement of nucleic acids present in solution. One need only count the number of a given type of sequences present to determine it’s abundance. Unlike DNA arrays, sequencing is not dependent on prior knowledge of which nucleic acids may be present.

43. The Future of DNA arrays Sequencing is also able to independently detect closely related gene sequences, novel splice forms or RNA editing that may be missed due to cross hybridization on DNA microarrays As a result of these advantages and the decreasing cost of sequencing, DNA arrays are being rapidly replaced by sequencing for nearly every assay

44. Take home message Microarrays are a powerful tool and holds much promise for the analysis of diseases. Classifications of disease by DNA, RNA, or protein profiles will greatly enhance our ability to diagnose, prevent, monitor and treat our patients. Microarrays promise a more biologically based, individualized treatment

45. Thank You