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Preparation of Sample Hybridization Scanning and Image Analysis

Preparation of Sample Hybridization Scanning and Image Analysis. 1. Design experiment. Question? Replicates? Test?. mutant. wild type. 2. Perform experiment. 3. Precipitate RNA. Eukaryote/prokaryote? Cell wall?. 4. Label RNA. Amplification? Direct or indirect? Label?.

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Preparation of Sample Hybridization Scanning and Image Analysis

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  1. Preparation of Sample Hybridization Scanning and Image Analysis

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

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

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

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

  7. TTTTTTTTT TTTTTTTTT Sample preparation • Combine • Hybridize

  8. Hybridization

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

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

  13. Hybridization Probe length

  14. Scanning Images Microarray technology Washing

  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 Excitation Emission Flourescence

  16. Excitation Emission Excitation Emission Cyanine Dye spectra excitation and emission

  17. Alexa Dyes comparison of excitation spectra

  18. Confocal scanner diagram

  19. The confocal scanner scans the slide

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

  21. Microarray image formats • 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 Cy 3 Channel Cy 5 Channel

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

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

  24. Density Intensity Spot Segmentation Overview

  25. Fixed Circle segmentation • 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

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

  27. Adaptive Shape segmentation 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.

  28. Density Intensity Histogram segmentation • 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

  29. Background Estimation Spot ScanAlyze QuantArray examples

  30. Spot Irregularity examples 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

  31. Quality Measures examples • 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

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

  33. Output Example (ImaGene) Field Meta Row Meta Column Row Column Gene_ID Flag Signal MeanBackgroundMean A 1 1 1 2 ZY030076 0 4655 463 A 1 1 1 3 ZY030066 0 15938 405 A 1 1 1 4 ZY029209 0 7441 390 A 1 1 1 5 ZY030089 0 1842 399 A 1 1 1 6 ZY030084 0 6864 401 A 1 1 1 7 ZY007003 2 471 481 A 1 1 1 8 ZY006869 0 8576 447 A 1 1 1 9 ZY007954 0 4965 405 A 1 1 1 10 ZY006866 0 2236 374 A 1 1 1 11 ZY006782 0 2088 355 A 1 1 1 12 ZY006907 0 4726 342 A 1 1 1 13 ZY006593 0 4437 338 A 1 1 1 14 ZY006850 0 917 321

  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

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