Protocol for Analysis of Mouse Membrane Array Data
Downloads:
The
following downloads provide analysis tools for processing mouse membrane array
data in a Windows-PC environment using Microsoft Office tools. The macros
embedded in these Microsoft Excel workbooks were designed to work with Clontech Atlas Mouse 1.2 arrays. The macros
are written in Visual Basic and can be modified in VB Editor for use with
other array designs.
Statistical Considerations:
We routinely obtain 3 replicates of each experimental condition when using
the Clontech Atlas arrays (single spots for each gene). The uncertainty
that lies behind individual gene measurements can be variously calculated
as the standard deviation of the determinations or the coefficient
of variation can be used. Since we are usually interested in the
statistical significance of differences between an experimental
condition and a control, we prefer the student t-test as a means
for calculating the probability, based on the uncertainty of the
measurements in both conditions, that gene expression differs significantly
between the conditions. We use the standard deviation of the mean
of the log ratios, within the context of the P value, to indicate
significant up- or down-regulation of gene expression. This approach
is meaningful where the expression level of the majority of genes
does not change significantly between conditions and where the researcher
is interested in genes that show substantially different expression.
The standard deviation for the log ratios is calculated and only
those genes that differ by more than 3 standard deviations (99.9%
confidence in each tail) from the mean of the log ratio (usually
zero, or no change) are considered.
Array Data Analysis in Excel:
The first VB macro, (embedded in MouseCruncher.xls) assigns the gene name to
each spot by its coordinate (location) on the array and normalizes
individual hybridization replicates by expressing the signal intensity
for each spot as a percentage of the sum of all spot intensities.
The second macro (embedded in MouseAnalysis.xls) streamlines statistical analysis
of experimental replicates by application of the student t-test
to calculate p-values that indicate confidence levels for the significance
of the ratio of experimentals to controls, based on the uncertainty
of individual genes in the 3 replicates, introduces a threshold
value, calculates expression ratios between experimental and control
conditions, and calculates an additional statistical metric, the
standard deviation of the mean of the log ratios. Step-by-step instructions
follow:
Open
the workbook called "MouseCruncher", being sure to enable macros
and open as read-only. [This workbook has two worksheets, named:
ARVDATA and DataAnalysis. The "ARVDATA" worksheet is
blank and the "data-analysis" worksheet contains annotation
information to assign the gene name to each spot on the image.]
Open the .XLS file that contains the exported raw data from Array
Vision. Select colums A-C, and copy/paste this information into
the blank "ARVDATA" worksheet of the "MouseCruncher"
workbook beginning at cell A1. To run the macro, choose Tools
on the toolbar, select Macros, highlight "allanalysis3"
and click Run. Save the crunched data as "condition-replicate-crunched"
following your standardized file-naming scheme.
The second workbook (MouseAnalysis) is used to execute the student
t-test on two replicate data sets for two conditions and calculates
the log ratio and the standard deviation of the mean of the log
ratios. [The MouseAnalysis workbook has four worksheets named:
"enterdata", "PRAW", "PLN",and "AllAnalysis".]
Open the 6 "condition-replicate-crunched" files (3 each
for control and experimental conditions) and the "MouseAnalysis"
workbook. The 6 data sets are sequentially copied and pasted beginning
with the first replicate of the control condition followed by the
second and third replicates of the control, then the first, second,
and third replicates of the experimental. Select columns A-C of
the crunched data set in the "DataAnalysis" worksheet
in the "condition-replicate-crunched" and copy/paste into
the corresponding 3 columns of the "enterdata" worksheet
in the "MouseAnalysis" workbook at the appropriate cell
on row 1. The process is repeated for the remaining 5 replicates.
To run the macro, choose Tools, select Macros, "AllAnalysis9",
and click Run. Save the file as "condition-replicate-analysis",
following your file-naming scheme.
Workbooks and Macros
Workbook |
Macro |
Subroutine |
Purpose |
MouseCruncher
|
allanalysis3
|
copy1 |
copies
and pastes data from ARVDATA to DataAnalysis worksheets |
MouseCruncher
|
allanalysis3
|
calcpctl2 |
normalizes
data by expressing each spot as percentage of sum of all
spot intensities |
Workbook |
Macro |
Subroutine |
Purpose |
MouseAnalysis
|
AllAnalysis9
|
spotsort1 |
sorts
the 6 data sets individually by spot number |
MouseAnalysis
|
AllAnalysis9
|
CalcAvg2 |
calculates
averages of volumes and pct values for the control and test
replicates |
MouseAnalysis
|
AllAnalysis9
|
copyvaluesintoPRaw3 |
copies
and pastes pct values into a separate spreadsheet for calculation
of p values |
MouseAnalysis
|
AllAnalysis9
|
CalcPRaw4 |
calculates
the Pvalue for the raw data by application of the student
t-test |
MouseAnalysis
|
AllAnalysis9
|
CalcLn5 |
copies
pct values into a separate spreadsheet and natural log transforms
data |
MouseAnalysis
|
AllAnalysis9
|
CalcPLn6 |
calculates
the Pvalue for the log transformed data by application of
the student t-test |
MouseAnalysis
|
AllAnalysis9
|
CopyValuesintoAnalysis7 |
copies
and pastes data used for ratio calculations into separate
spreadsheet |
MouseAnalysis
|
AllAnalysis9
|
CalculateRatios8 |
calculates ratio of Test/Control,
reorganizes data, and calculates log (10) of ratio |
Directory Structure:
Array Analysis
User Name
Project
Name
Images
(.gel TIFF image files)
Export
(.xls raw data files from ArrayVision)
Crunched
(.xls crunched image files created in "MouseCruncher")
Analysis
(.xls files with ratio calculations created in “MouseAnalysis”)
|