Preparation of Sample Hybridization Scanning and Image Analysis
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Transcript of Preparation of Sample Hybridization Scanning and Image Analysis
Preparation of Sample Preparation of Sample
HybridizationHybridization
Scanning and Image Scanning and Image AnalysisAnalysis
Sample preparation
1. Design experimentQuestion?Replicates?Test?
2. Perform experiment
4. Label RNAAmplification?Direct or indirect?Label?
wild typemutant
3. Precipitate RNAEukaryote/prokaryote?Cell wall?
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
Sample preparation
AAAAAAAAARNA
TTTTTTTTT
aa-dUTP
cDNA
• Revese Transcriptase (RT)• oligo dT primer• nucleotides (dNTP)• amino-allyl-dUTP (aa-dUTP)
Sample preparation
AAAAAAAAARNA
TTTTTTTTTcDNA
• Hydrolysis (NaOH and EDTA Tris)• Clean-up
Sample preparation
TTTTTTTTTcDNA
• Coupling (Cy3 or Cy5)• Quenching• Clean-up
Sample preparation
TTTTTTTTT
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 C2 h
16 C2 h
37 C6 h
70 C10 min
Detection of Biotin (Affymetrix)
Streptavidin Phycoerythrim = SAPE ( )
anti-SAPE IgG
biotinylated anti-anti IgG
Materials and Methods
All protocols can be found here:
http://cmgm.stanford.edu/pbrown
and here:
http://www.affymetrix.com
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
HybridizationProbe length
Scanning Images
technology
Washing
Microarray
Labeling dyesand 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
Cyanine Dye spectra
Excitation Emission Excitation Emission
excitation and emission
Alexa Dyescomparison of excitation spectra
Confocal scannerdiagram
The confocal scannerscans 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 0-65535
The image resolution is commonly 10m [currently, max 5m]
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 percentileForeground : 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 Irregularityexamples
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 Intensitycalculation
The average or median pixel value in the spot and background masks are calculated.
Output
Field Meta Row Meta Column Row Column Gene_ID Flag Signal Mean Background Mean
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
Example (ImaGene)
Examples of Difficult Imagesmicroarray 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: 1859960073
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