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

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aa-dUTP

cDNA

• Revese Transcriptase (RT)• oligo dT primer• nucleotides (dNTP)• amino-allyl-dUTP (aa-dUTP)

Sample preparation

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TTTTTTTTTcDNA

• Hydrolysis (NaOH and EDTA Tris)• Clean-up

Sample preparation

TTTTTTTTTcDNA

• Coupling (Cy3 or Cy5)• Quenching• Clean-up

Sample preparation

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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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