MACALCULATE procedure

Corrects and transforms two-colour microarray differential expressions (D.B. Baird).


Options

PRINT = string
What to print (summary); default summ

BMETHOD = string
How to correct for spot foreground for background values (subtract, smooth, none); default subtracts REDBACKGROUND and GREENBACKGROUND if set

TRANSFORMATION = string
Type of transformation to apply to the red/green ratios (log, glog); default log

MINIMUM = scalar
Minimum value per channel; if RSDBACKGROUND and GSDBACKGROUND are supplied, this is the multiplier of these per spot, default 0

PERSPOTMINIMUM = string
Use a single minimum value per spot rather than per slide (yes, no); default no

CONSTANTVALUE = scalar
Constant to add to red and green foreground values; default 0

DF = scalar
Degrees of freedom to use for loess smoothing of background; default 20


Parameters

RFOREGROUND = variates
Red foreground values per spot

GFOREGROUND = variates
Green foreground values per spot

RBACKGROUND = variates
Red background values per spot

GBACKGROUND = variates
Green background values per spot

RSDBACKGROUND = variates
Standard deviation of Red background

GSDBACKGROUND = variates
Standard deviation of Green background

SLIDES = factors
Defines the slide to which each spot belongs for smoothing, or per slide minimums

ROWS = factors
Defines the row position of each spot for background smoothing

COLUMNS = factors
Defines the column position of each spot for background smoothing

LOGRATIOS = variates
Saves the differential expression per spot

INTENSITIES = variates
Saves the intensity of each spot


Description

MACALCULATE calculates log-ratios from a two channel microarray. There are options for background correction and to set the channels to a minimum value to avoid large variation when the values in either channels get close to zero. The ratios are logged as the variation usually increases with the size of the mean. However, although the variation may be more uniform with the logged ratios, it may still show considerable heterogeneity with different levels of intensity.

   The REDFOREGROUND and GREENFOREGROUND parameters supplies variates containing the values of the red/Cy5 and green/Cy3 foreground for each spot (Cy5 and Cy3 are the technical names for the red and green fluorescent dyes). The SLIDES parameter supplies a factor indicating the slide from which each spot was read.

   The BMETHOD option controls the method to use for background correction, with settings:

    none
no corrections;

    subtract
subtract the red and green backgrounds, which must then be supplied by the REDBACKGROUND and GREENBACKGROUND parameters;

    smooth
perform a two-dimensional loess smoothing of the background values, and uses these for the correction: the ROWS and COLUMNS parameters must then supply factors to define the row and column positions of each spot.

If BMETHOD is unset, the red and green backgrounds are subtracted if available. The DF option specifies the number of degrees of freedom for the smoothing; if this is unset, the default is to use the square root of the number of spots observer per slide. Note that smoothing can be time consuming for large slides.

   Log-ratios are undefined where spots may have foreground levels that are below their background levels, as you cannot take the log of a negative number. If this happens for both red and green, then there is no valid information on the level of differential expression and the log-ratio must be set to a missing value. However, there may be some useful information when one channel is above background and the other below and, if these log-ratios too are set to missing values, probes with a high level of differential expression may be missed. Some image analysis packages set a very large constant log-ratio for this, but this can bias the results as no differentiation is made between cases where the other colour is just above background, and other where the other colour is significantly above background.

   You can set the MINIMUM option to a positive value to apply minimum values to the colours (for the default value of zero, no minimum values are applied). In the simplest situation, any red or green value less than MINIMUM is set to MINIMUM. For the alternatives, you need to supply variates giving the standard deviations of the red and green backgrounds around each spot, using the RSDBACKGROUND and GSDBACKGROUND parameters. Then, if option PERSPOTMINIMUM=yes, the background standard deviation for each colour is multiplied by MINIMUM, so that the minimum value depends on the quality of the background around each spot. With the default, PERSPOTMINIMUM=no, MINIMUM is multiplied by the background standard deviation for each colour, averaged within each slide.

   The CONSTANTVALUE option can be used to reduce variability at the low end of the intensity range, as often the ratios become unstable as the foreground gets close to the background level. Adding a constant to both red and green will stabilize the log-ratios at low intensities, but with a potential loss of sensitivity for detecting differential expression. CONSTANTVALUE would normally be a positive value, although a negative value could be used if it were thought that the image analysis package was underestimating the background values. By default CONSTANTVALUE=0.

   The LOGRATIOS parameter can supply a variate to save the calculated log-ratio or generalized log-ratio transformation (as requested by the TRANSFORMATION option) of the red and green values for each spot. The INTENSITIES parameter can supply a variate to save the intensity of each spot:

Intensity = (Log(Red) + Log(Green)) / (2 × Log(2))

 

Options: PRINT, BMETHOD, TRANSFORMATION, MINIMUM, PERSPOTMINIMUM, CONSTANTVALUE, DF.

Parameters: RFOREGROUND, GFOREGROUND, RBACKGROUND, GBACKGROUND, RSDBACKGROUND, GSDBACKGROUND, SLIDES, ROWS, COLUMNS, LOGRATIOS, INTENSITIES.