Upon choosing and starting to use a microarray platform, several experimental parameters need to be optimized before performing an experiment. Microarray manufacturers optimize appropriate conditions specific to their platform such as hybridization time, temperature and washing stringency. In a microarray experiment, the relative levels of gene expression are directly proportional to the intensity of a hybridization signal. High hybridization intensity at a given microarray spot indicates that the original sample contains a relatively high abundance of the corresponding transcript. Low hybridization intensity means the transcript is relatively rare.
The hybridization reaction consists of two substrates: the nucleic acid on the array and the labeled nucleic acid in solution. The researcher controls the amount of labeled sample incubated with the array. The amount of labeled sample used in a microarray experiment depends on balancing two opposing considerations.
1. The hybridization of more labeled sample to the microarray maximizes the detection of less abundantly expressed genes, increases the present call, and decreases the number of false negatives.
2. Increasing the amount of the labeled sample causes signal saturation, decreases the ability to resolve changes in the expression of more-abundantly expressed genes, and increases the number of false positives.