A control gene that is expressed at a constant level is used to normalize the gene expression results for variable template amount or template quality. If RNA quantitation can be done accurately, normalization might be done using total RNA amount used in the reaction. The use of multiple housekeeping genes that are most appropriate for the target cell or tissue is the most optimal means for normalization. This normalization is performed by the experimenter and should not be mixed up with the normalization of fluorescence signal using the passive reference dye (usually ROX) performed by the equipment. See Normalization Methods for qPCR; Dheda, 2004.