Microarray dye balance - Slide preparation, probe labeling, hybridization (Jan/14/2005 )

Dear Colleagues,

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.

Simcerely,

Dr. Igor Kostenyuk,
University of Florida,
Lake Alfred, FL 33884

Hello

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.


dnamicro
http://www.dnamicroarrays.info

Igor,

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!

Good luck,
SV

wow and i thought it was just me!

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.

i've tried for ages and got it to work....

we excluded our ethanol steps in the washing and the signal intensities are quit good and balanced. the amersham cy5 thus seems to be quenced enourmously by ethanol

I got similar problem.

I can't balance Cy3 and Cy5 at all. Cy3 is way higher than Cy5. I was wondering that if it's due to sample's treatment. However, it sounds unreasonable that treatment will lead to most of the genes downregulated.

Now, I am thinking about dyes too

DML, you mentioned you exluded the ethanl part when washing. can you tell more detail? do you mean washing of slides? we acutally did not use ethonal to wash slides, but use isopropanol to wash pre-hybridized slide. does it also matter?

thanks a lot,

forforfor

one more thing, I got 0.7 pmol of cy3 and 0.5 pmol of cy5. if it is a reason of unbalanced dyes, how could I get equal labelling?


thanks

forforfor

Dear Igor ,
First of all before starting hybridization experiment you check spec. reading of labelled
cDNA with Cy3/Cy5 and also the quality of labelled cDNA by runing on the 1% agrose gel (which is cast on the slide very thin) after runing unpurified and labelled cDNA you scan its on the microarray scanner or on the other scanner.
After geting good result you finally take equal pmole amount for hybridisation on the chip( In our case we are using torento slides which gives good result after hybridising with 40 pmole of each dye labelled cDNA.
Best of luck.
awadh