Microarray dye balance
Posted 14 January 2005 - 02:00 PM
Could anybody tell me how to improve color balance in
Microarray labeling/hybridization business and enhance
red color since my image is preferably green regardless of
what color is Control or Experimental Sample was labeled with?
Suggestions/ideas are highly appreciated.
Dr. Igor Kostenyuk,
University of Florida,
Lake Alfred, FL 33884
Posted 17 January 2005 - 06:16 AM
First check your labelling efficiency by spec and try to use equal amount of pico moles for both the dyes. Also check the specific activity of the dyes, if there is a lot of difference between specific activity of both the dyes then also it happens. it may be possible that your red dye may be a problem.
Posted 30 January 2005 - 09:22 PM
Are you using Amersham's Cy5?!! Dump it! I wasted hundreds of USD and almost lost my senses trying to get the dye to work for months. Then I saw several comments from people (frustrated users of Amersham's Cy5) who were unhappy with the dye. In my case, switching to PerkinElmer's Cy5 did wonders! I have no complaints about Amersham's Cy3 though, works great!
Posted 31 January 2005 - 02:34 PM
i tried for AGES to get Amersham Cy5 to work, and people thought i was mad when i said it didnt. i called amersham on numerous occasions and they kept telling me that i was the only one that had reported the problem - although in all fairness they did replace the dyes twice. Cy3 has always worked for me though.
I ended up switching to AlexaFluor dyes (molecular probes - they sent me some free samples to try first). But i also found adding DTT to the washes helped a bit. i would also try and quantitate the labelling reaction (desribed in the amersham manual). i was doing in-direct labelling with amino-allyl-dUTP, so i dont know if direct labelling would work differently. also, i found that if you labelled more cDNA than the recommended amount ( i was using both amplified RNA and total RNA - around 10 micrograms of amplified RNA or 40 micrograms of total RNA. probably a bit high but it gave good results) the Cy5 seemed to work better.
so i cant make any sense of it. hopefully these suggestions help.