A researcher a friend of mine send me quality control report of result of one sample cyto CGH microarray using Agilent slides 4 x 180. I notes that some quality control parameters indicating that is some problems in this analysis for example: the signal of histogram of plot signals in the red color and green color is quite different, the red signals give 2200 “number of probes” while the green give 3000 “number of probes”. Another issue the signal intensity give only 30 but it should give something between 200-400, I think that the problem may be in the fluorescent labeling ,in probe degradation or fluorescent quenching
I much appreciate your help if someone who is working in this microarray system to do reanalysis and let me know what is the kind of error that may be concluded from the analysis.
Dear Sajedah,
it is helpful to determine the incorporation rates prior to hybridization using a Nanodrop photometer. Was this done and if so, how were the results?
Dear Torsten Goldmann,
Thanks for your answer, please find the attached Nanodrop photometer report to give you idea about the activity and incorporation rate after labeling, I hope it will be helpful to complete the answer, I much appreciate your efforts and your willingness to help
waiting for answer
sajedah
Dear Sajedah,
For me the specific activity of your labelling (if it is in pmol dye/microgram cDNA) seems to be more or less OK. Of course, the higher the better, about 50-100 is great, but we have done good hybridisations with similar specific activity to yours. So, other issues has to be considered. Strictly follow Agilent's hybridisation and washinf protocols, and minimize light exposure of the labelled cDNA, for axample by using black or amber coloured eppendorf tubes. The raw fluorescence reads should be in the range of 10 to 10000, so even 30 can be fine if it is not for every spots.
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