Sorry for the formatting error.
Mine is a two color experiment, where each of four chips is a
biological
replicate, and half of these are dye-swapped. The (biological) control
is
always in the first channel and the treatment is always in the other.
My
confusion must stem from the R/G mnenomic: Cy5 fluorescence is in the
red
and Cy3 in the green, but this is irrelevant. Limma's R/G refer to
false
color. My controls are always false colored red, and the experimentals
always green, irrespective of the dye. Thus, all of the control values
should go into one matrix, and all the experimental values into the
other,
and my design would be c(1,1,1,1). Half of the measurements in the R
channel
should be cy3 measurements and half should be cy5 measurements. That
way,
dye-biased measurements from the same spot, assuming no biological
change,
should average out to zero, after normalization. Does that sound
right?
John Welsh
Associate Professor
Sidney Kimmel Cancer Center
10835 Altman Row
San Diego, CA 92121
(858) 450-5990 ex.282
jwelsh@skcc.org
At 08:36 AM 13/12/2003, John Welsh wrote:
>Sorry for the formatting error.
>
>Mine is a two color experiment, where each of four chips is a
biological
>replicate, and half of these are dye-swapped. The (biological)
control is
>always in the first channel and the treatment is always in the other.
My
>confusion must stem from the R/G mnenomic: Cy5 fluorescence is in the
red
>and Cy3 in the green, but this is irrelevant. Limma's R/G refer to
false
>color.
Limma's R/G corresponds to Cy5/Cy3 dyes where that information is
available, for example with Genepix data. Otherwise "green" is a
synonym
for channel 1 and "red" is a synonym for channel 2. (If you break the
convention that Cy3=channel 1 and Cy5=channel 2, then it's up to you
to
unravel it.)
Gordon
> My controls are always false colored red, and the experimentals
>always green, irrespective of the dye. Thus, all of the control
values
>should go into one matrix, and all the experimental values into the
other,
>and my design would be c(1,1,1,1). Half of the measurements in the R
channel
>should be cy3 measurements and half should be cy5 measurements. That
way,
>dye-biased measurements from the same spot, assuming no biological
change,
>should average out to zero, after normalization. Does that sound
right?
>
>John Welsh
>Associate Professor
>Sidney Kimmel Cancer Center
>10835 Altman Row
>San Diego, CA 92121
>(858) 450-5990 ex.282
>jwelsh@skcc.org
