Once the microarray hybridization protocol has been carried out and the slides have been scanned, the next step is to quantify the images. Scanning produces two .tif image files. One represents the test readings and one the control readings. Both of these image files are loaded together into BlueFuse for quantification of each spot.
BlueFuse has been developed by a Cambridge based company called BlueGnome.
It uses Bayesian algorithms to generate intensity values for each spot
on the array. Briefly, the Cy5 and Cy3 files are loaded into the software
and are overlaid onto one another. BlueFuse uses a .Gal file for each
batch of arrays. This .Gal file contains array information for each gene
and its position on the array. The software also allows positive and negative
controls to be recorded. Once these have been set the software is ready
BlueFuse also offers users to 'fuse' their quantified data. This combines
spots from the same array on the basis of the estimated confidence in
each ratio and the level of agreement between replicate spots. Once optimization
is complete, quantification is automatically carried out via the software.
The results are saved as .txt files and the files can now be loaded into
an analysis analysis.
Recent advances in BlueFuse include the ability to carry out normalization
and additional downstream analysis processes. With its ease of use and
lack of user intervention, BlueFuse offers a good overall package for
To view a basic protocol for BlueFuse, click here.
To view frequently asked questions on BlueFuse, click here.