ReviewChanged Relative to What? Housekeeping Genes and Normalization Strategies in Human Brain Gene Expression Studies
Section snippets
Why Normalize?
Why normalize and what is hoped to be achieved by doing so? These are basic yet deceptively complex questions. A typical explanation (14) is that “several variables need to be controlled for in gene expression analysis, such as the amount of starting material, enzymatic efficiencies, and differences between tissues or cells in overall transcriptional activity.” This quotation highlights that there are a number of factors that contribute to variability in a gene expression dataset. Accordingly,
Normalization to What?
Once it has been decided that normalization is required, the question becomes: normalization to what? Although normalization to endogenous control genes is the most commonly used approach in studies of gene expression as a whole and is almost always adopted in postmortem brain studies, it is not the only option. An alternative method sometimes advocated is to use an exogenously applied transcript, that is, to “spike” the sample with an external RNA standard. However, this does nothing to
Conclusions and Recommendations
Normalization should remain an integral part of quantitative human brain gene expression studies because, despite the various caveats and complexities discussed here, it is always advantageous and often critical. However, appropriate flexibility in normalization strategies should be allowed—even encouraged—because there is no perfect or universally applicable way to normalize: “commentators who assert there is a correct and definitive way to quantify mRNA fail to understand the complexities” (57
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