2. Sample preparation 1. Design experiment Question? Replicates? Test? 2. Perform experiment 4. Label RNA Amplification? Direct or indirect? Label? wild type mutant 3. Precipitate RNA Eukaryote/prokaryote? Cell wall?

3. Sample Preparation - Check quality of RNA Load 50-500 ng of RNA on the BioAnalyzer eukaryotic: 28S/18S ≈ 2.0 prokaryotic: 23S/16S ≈ 2.0

4. Sample preparation AAAAAAAAA RNA TTTTTTTTT aa-dUTP cDNA Revese Transcriptase (RT) oligo dT primer nucleotides (dNTP) amino-allyl-dUTP (aa-dUTP)

5. Sample preparation AAAAAAAAA RNA TTTTTTTTT cDNA Hydrolysis (NaOH and EDTA  Tris) Clean-up

6. Sample preparation TTTTTTTTT cDNA Coupling (Cy3 or Cy5) Quenching Clean-up

7. Sample preparation TTTTTTTTT Combine Hybridize

8. Hybridization

9. Sample Preparation - Eberwine RNA T7 dsDNA T7 pol SAMPLE ssDNA + Reverse Transcriptase + RNase H + Polymerase clean up dsDNA + Biotin-labeled nucleotides aRNA 42  C 2 h 16  C 2 h 37  C 6 h 70  C 10 min

10. Detection of Biotin (Affymetrix) Streptavidin Phycoerythrim = SAPE ( ) anti-SAPE IgG biotinylated anti-anti IgG

11. Materials and Methods All protocols can be found here: http://cmgm.stanford.edu/pbrown and here: http://www.affymetrix.com

12. Scanning and image analysis Scanning -Dyes -Confocal scanner -CCD scanner Image File Formats Image analysis -Locating the spots -Segmentation -Evaluating data quality by H. Bjørn Nielsen

13. Hybridization Probe length

14. Scanning Images technology Washing Microarray

15. Labeling dyes and their properties The two most common fluorochromes used are: Cyanine3 (cy3, exicitation = 554, emission = 568) Cyanine5 (cy5, exicitation = 650, emission = 672) But Alexa dyes are also becoming popular ExcitationEmission Flourescence

16. Cyanine Dye spectra ExcitationEmissionExcitationEmission excitation and emission

17. Alexa Dyes comparison of excitation spectra

18. Confocal scanner diagram

19. The confocal scanner scans the slide

20. CCD scanner CCD camera Emission filter Excitation filter Beamsplitter White light detects from an area

21. Microarray The most common file format is 16bit TIFF. A 16bit TIFF file describe each pixel in an image with an intensity ranging from 0-65535 The image resolution is commonly 10  m [currently, max 5  m] Normally two scans in different wavelengths result in two monochrome files that are overlaid Pseudo-color overlay image formats Cy 3 Channel Cy 5 Channel

22. Image analysis Locating the spots Segmentation Ensuring good data quality (flagging) Density Intensity overview

23. Locating A grid is laid over the image to aid the program in identifying the individual spots Most programs have some automation in this step. the spot features

24. Spot Segmentation Fixed circle ScanAlyze, GenePix, QuantArray Adaptive circle GenePix, Dapple Adaptive shape Spot, (a R package) Histogram method ImaGene, QuantArray DeArray Density Intensity Overview

25. Fixed Circle Fits a circle with a constant diameter to all spots in the image Easy to implement The spots need to be of the same shape and size segmentation

26. Adaptive Circle The circle diameter is estimated separately for each spot Problematic if spot exhibits non-circular shapes segmentation

27. Adaptive Shape Starts by Specifying a starting points (given by the gridding) Regions grow outwards from the starting point according to the difference between a pixel’s value and the running mean of values in an adjoining region. segmentation

28. Histogram Density Intensity Uses a target mask chosen to be larger than any spot Foreground and background intensity are determined from the histogram of pixel values for pixels within the masked area Example : Background : mean between 5th and 20th percentile Foreground : mean between 80th and 95th percentile segmentation

29. Background Estimation QuantArray ScanAlyze Spot examples

30. Some spots may be more uncertain than others. This can be caused by: -Dust grains -Background smear -Strange shaped spot (comet tails, etc.) -Donuts shaped spots -Weak signals Spot Irregularity examples

31. Quality Measures We can pickup most of these irregularities by these measures - Intensity variability measures - Spot size deviation - Circularity deviation - Relative signal to background intensity - Position deviates from a rectangular grid Based on such measurements, a spot can be rejected examples

32. Feature Intensity calculation The average or median pixel value in the spot and background masks are calculated.

33. Output FieldMeta RowMeta ColumnRowColumnGene_IDFlagSignal Mean Background Mean A1112ZY03007604655463 A1113ZY030066015938405 A1114ZY02920907441390 A1115ZY03008901842399 A1116ZY03008406864401 A1117ZY0070032471481 A1118ZY00686908576447 A1119ZY00795404965405 A11110ZY00686602236374 A11111ZY00678202088355 A11112ZY00690704726342 A11113ZY00659304437338 A11114ZY006850 0917321 Example (ImaGene)

34. Examples of Difficult Images microarray images

35. Book about hybridization Nucleic Acid Hybridization (Introduction to Biotechniques S.) By: M.L.M. Anderson Paperback 256 pages (December 1998) Publisher: BIOS Scientific Publishers ISBN: 1859960073