Sample preparation 1. Design experiment Question? Replicates? Test? 2. Perform experiment 4. Label RNA Amplification? Direct or indirect? Label? wild.

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Presentation transcript:

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?

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

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

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

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

Sample preparation TTTTTTTTT Combine Hybridize

Hybridization

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

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

Materials and Methods All protocols can be found here: and here:

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

Hybridization Probe length

Scanning Images technology Washing Microarray

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

Cyanine Dye spectra ExcitationEmissionExcitationEmission excitation and emission

Alexa Dyes comparison of excitation spectra

Confocal scanner diagram

The confocal scanner scans the slide

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

Microarray The most common file format is 16bit TIFF. A 16bit TIFF file describe each pixel in an image with an intensity ranging from 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

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

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

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

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

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

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

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

Background Estimation QuantArray ScanAlyze Spot 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 Spot Irregularity examples

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

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

Output FieldMeta RowMeta ColumnRowColumnGene_IDFlagSignal Mean Background Mean A1112ZY A1113ZY A1114ZY A1115ZY A1116ZY A1117ZY A1118ZY A1119ZY A11110ZY A11111ZY A11112ZY A11113ZY A11114ZY Example (ImaGene)

Examples of Difficult Images microarray images

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: