1. DNA Microarray Overview and Application

2. Table of Contents Section One : Introduction Section Two : Microarray Technique Section Three : Types of DNA Microarrays Section Four : Application of DNA Microarrays 2

3. Introduction Section One

4. Era Of Genome 4 Can be used as diagnostic profile Example: cancer diagnosis Can show how RNA levels change during development, after exposure to stimulus, during cell cycle, etc. Can help us start to understand how whole systems function Provides large amounts of data RNAPROTEIN DNA TRANSCRIPTION TRANSLATION S-1

5. Nucleic Acid Hybridization 5 S-1 Hybridization is Used to Measure Gene Expression

6. Blotting Techniques 6 S-1

7. Solution for mass of information 7 S-1 Microarray

8. DNA Microarray: Definition 8 S-1 DNA microarrays are solid substrates on which thousands of DNA molecules corresponding to the genes under investigation are deposited in an ordered arrangement. DNA Microarrays consist of 100 - 1 million DNA probes attached to a surface of 1 cm by 1 cm(chip).

9. DNA Microarray: Definition 9 S-1 By hybridization, they can detect DNA or RNA:

10. Microarray Vs. Blotting These arrays can carry out studies that were previously done using standard techniques of molecular biology (Northern or Southern blots), but with two major differences: The known genomic sequence is immobilised on the solid substrate. The system is mini-aturised in such a way that many probes can be integrated in parallel. 10 S-1

11. Microarray Technique Section Two

12. DNA Microarray 1. Collect Samples. 2. Isolate mRNA. 3. Create Labelled DNA. 4. Hybridization. 5. Microarray Scanner. 6. Analyze Data. 12 S-2

13. Isolate mRNA Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform. After isolating the RNA, we need to isolate the mRNA from the rRNA and tRNA. mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind the mRNA. Rinse with buffer to release the mRNA from the beads. The buffer disrupts the pH, disrupting the hybrid bonds. 13 S-2

14. DNA Microarray : Labelled DNA  Add a labelling mix to the RNA. The labelling mix contains poly-T (oligo dT) primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides.  We will add cyanine 3 (fluoresces green) and cyanine 5 (fluoresces red).  The primer and RT bind to the mRNA first, then add the fluorescently dyed nucleotides, creating a complementary strand of DNA 14 S-2

15. DNA Microarray : Hybridization 15 S-2

16. DNA Microarray : Scanner  The scanner has a laser, a computer, and a camera.  The laser causes the hybrid bonds to fluoresce.  The camera records the images produced when the laser scans the plate.  The computer allows us to immediately view our results and it also stores our data. 16 S-2

17. DNA Microarray : Scanner 17 S-2

18. Types of DNA Microarray Section Three

19. Types of Microarrays Depending on how they have been manufactured : Spotted Synthesized Self-assembled Depending on the application that microarrays are designed for : expression microarrays microRNA arrays protein microarrays Gene expression arrays : two-channel one-channel 19 S-3

20. Spotted Microarrays Probes are first synthesized and then printed, or “spotted” on a glass slide. Sequences for the probes are obtained from public information. Probes can be either long polynucleotides or shorter oligonucleotides. 20 S-3

21. Spotted Microarrays 21 S-3

22. Spotted Microarrays 22 S-3

23. In Situ Synthesized Microarrays cross-hybridization problems due to the length of the probes in Spotted Microarrays. As an alternative, the company Affymetrix developed a new type of microarrays. Affymetrix probes are synthesized directly on the support using a process called Photolithography. Affymetrix : www.affymetrix.com NimbleGen : www.nimblegen.com Agilent :www.agilent.com 23 S-3

24. Affymetrix technology 24 S-3

25. Affymetrix 25 S-3

26. Affymetrix technology 26 S-3

27. Affymetrix technology 27 S-3

28. Affymetrix : GenChip 28 S-3

29. NimbleGen technology 29 S-3

30. Agilent technology 30 S-3

31. Agilent 31 S-3

32. Self-assembled Microarrays 32 S-3

33. Self-assembled Microarrays 33 S-3

34. Application of Microarrays Section Four

35. Gene Expression Analysis Gene expression microarrays are used to measure gene expression levels by indirectly identifying and quantifying the mRNA transcripts present in the cells in a given experimental condition. One-Channel Two-Channel 35

36. Two-Channel Two-channel microarrays are based on the competitive hybridization of two samples, each of which has been labeled with one different fluorescent dye for the probes in the microarray. Use two-color : Cy3, fluorescent in the green Cy5, fluorescent in the red 36

37. Two-Channel 37 S-2

38. Two-Channel 38 S-2

39. One-Channel Use one-color instead of two-color. Each RNA sample is analyzed in a separate microarray without relying on competitive hybridization. This approach provides absolute expression measures for each gene. 39

40. Measuring Gene Expression Gene expression quantification is performed through the analysis of the intensity of fluorescence signals intensity values are transformed into numerical values that will be submitted to bioinformatics processing and analysis. 40

41. Chip-on-Chip Arrays To study interactions between proteins and DNA sequences. The most common application of this type of arrays is the study of transcription factors. 41

42. DNA Microarray : An Experiment 42 S-4

43. References Alex Sa´nchez-Pla. 2014. DNA Microarrays Technology: Overview and Current Status. Comprehensive Analytical Chemistry, 63, pp 1-23. C. Nguyen and X. Gidrol. 2010. DNA Microarrays. Nanoscience, pp 1143-1161. Chris Seidel. 2008. Introduction to DNA Microarrays. Analysis of Microarray Data: A Network-Based Approach, pp 1-26. 43

44. Thanks Hossein Nassaj, Musa Mohammadi 44