To address these concerns, others have proposed normalizing target gene expression to the geometric mean of three stable Compound Library molecular weight reference genes. While equivalent directional shifts among three reference genes would be strongly indicative of technical variation supporting the use of DCT values exceeding 1.0, this method of normalization, which would be reagent and labor expensive, would still require validation to identify three references genes that do not display indices of biological CYT 11387 variance as marked deviation of even a single gene could significantly affect the geometric mean of multiple genes resulting in erroneous data interpretation. In some circumstances, the added expense may not be necessary as shown here and by others, where experimental outcome using a single, stable reference gene was equivalent to that observed when using the geometric mean of multiple, stable reference genes. It may also be necessary to validate more than three reference genes in order to identify at least three that are not affected by biological variance as shown in this report where validation of as many as six reference genes during adipocyte differentiation failed to identify three reference genes that displayed stable expression suitable for inclusion in geometric mean analyses. Therefore, it is imperative that all reference genes are validated for each experimental condition regardless of whether a single reference gene or the geometric mean of multiple reference genes is the chosen method of normalization. This premise should apply even when using established algorithms that quantify gene stability as variation between tissue and cell type or experimental treatment may lead to biological changes in gene expression that is otherwise stable or at least unaffected by biological variance. Albeit normalizing target gene expression to one or more reference genes is the method of choice of many investigators, it is important to note that some situations may warrant the evaluation of raw target gene expression that is not corrected for technical variance. As reference gene validation presents with its own set of difficulties, others have also proposed normalizing target gene expression to total RNA. However, neither method corrects for technical variation imposed by pipetting error, differences in PCR efficiency, or reaction interference imposed by contaminating reagents such as salts and phenol. It remains a matter of debate as to which method results in the most accurate experimental outcome. As one size may not fit all, any given technique may represent the best approach for a given set of circumstances or experimental conditions leaving the investigator to evaluate each method as warranted by the specific experiment under investigation.