AFGC

DNA Microarray Data Acquisition and

Analysis -

Introduction to Stanford Microarray

Database Damares C MonteCarnegie Institution of Washington,Plant Biology DepartmentStanford, CA

DNA Microarray Technology

Cy3: ~550 nmCy3: ~550 nmCy5: ~650 nmCy5: ~650 nm

No differential expressionNo differential expressionInducedInducedRepressedRepressed

AFGC

• Acquisition - QC

• Input/Storage/Retrieval

• Analysis/Pattern Recognition

• Visualization

• Interpretation/Annotation

• Publication/Repository

SMDSMD

Biologist

Data AnalysisData Analysis

Fluorescence Intensities Extraction

•To extract data from a microarray by accurately identifying the location of each of the spots.

•Data extracted on: Fluorescence intensities, background intensities, fluorescence intensities ratios.

•Load Images

•Adjust Gain (brightness of image) and Normalization (balance between images in red and green)

Create Grids

•Create new grid

•Number of grids to create

•Numbers of rows and columns

•Spot width and height

•Column and Row spacing

•Resolution (Xres and Yres)

•Tip spacing

•Create grid

SMD Overlay Cy3 Cy5

Dust speck

Saturated in both channels

Doughnut

Saturated on outside & in the middle doughnut

Saturated in one channel

Streak

Dust/Background/saturation/something coming into spot

What gets flagged?

Mean Intensity plots

Broad distribution pattern with curved or flattened arms. Common to have gap in the center. (Crab Claw)

Concentrated cluster of spots in a linear or fan pattern with clearly distinguished outliers

Median signal to background (How strong the signal is compared to background)Mean of median background (Determines how high is the background)Median signal to noise (Confidence to quantify peak signal to background) (F635Med - B635Med)/B635SD

Data Analysis FlowData Analysis FlowNew Scan

Gene Pix

Quality Control

Stanford Microarray Database - SMD

Data Selection

Quality Control

Complete Data Table (cdt)

Clustering

DownloadXML

Comparison plotThis plot can be effectively used to get a measure of the overall consistency between two duplicated or reversibly duplicated experiments. It compares the log2(RAT2N) values of the two experiments and calculates the regression line, which optimally should be close to one. Future functions will include filters and plotting of multiple experiments.

Why do we use log space?

Exponential distribution of intensitiesMost genes at low level Very few high level

Log scale approx. normal distribution??

Frequency of intensity levelsThis plot allows the user to plot the frequency of spots within

certain intensity intervals for the red and green channel respectively. It is possible to use normalized or non-normalized intensity values and linear or logarithmic scales. This graph will make it easier to determine background levels for filtering when using other analysis tools. The two examples below give a good

view of what normalization does to the data.

Frequency of intensity levels

Distribution of ratiosThis plot draws the distribution of the logarithm (base 2) of the red/green ratios [log2(RAT2N)]. It gives an immediate overview of the range of expression and the normality of the distribution.

Clustering and Image Generation Page

Spot Data PageSpot Data Page

Things to be Considered When Analyzing Microarray Data

1. Clones corresponding to spots with intensities of at least 350 in both channels and

ratios of 2.0 / 0.5 are worth further investigation.

2. For genes of interest, check that the spot(s) is acceptable.

3. Check for consistency among replicate values.

4. Validate important results obtained from the microarray data by an independent test or method.

@pilot

http://www.stat.berkeley.edu/users/terry/zarray/Html/log.html

AcknowledgementsAcknowledgementsShauna Somerville

Katrina Ramonell Lorne RoseBi-Huei HoSue ThayerShu-Hsing Wu

AcknowledgementsAcknowledgements

Shauna SomervilleShauna Somerville

Bench team

Katrina Ramonell

Lorne RoseBi-Huei HoSue ThayerShu-Hsing Wu

Bioinformatics team

David FinkelsteinJeremy GollubFredrik SterkyRob EwingLalitha SubramanianMira KaloperTodd RichmondMike Cherry

http://www.stat.berkeley.edu/users/terry/zarray/Html/log.html

http://www.stat.berkeley.edu/users/terry/zarray/Html/log.html