Normalization of microRNA quantitative RT-PCR data in reduced scale experimental designs.

Proper normalization of quantitative RT-PCR (qRT-PCR) data is a crucial component of gene -expression analysis. Although arbitrarily selected housekeeping genes have been used to normalize many published mRNA RT-PCR datasets, there is a growing awareness that such normalizers should be first validated empirically. The use of stable reference genes is particularly needed for qRT-PCR of microRNA (miRNA), which represent a novel class of biological regulators whose aberrant expression is associated with a range of disorders. Changes in miRNA levels can be modest, and yet have profound cellular consequences. As a result, precise measurements of miRNA expression are critically important. This chapter describes a detailed workflow for the selection of endogenous normalizers using the NormFinder algorithm, -resulting in more accurate miRNA expression profiling results. This approach is particularly well suited to smaller scale miRNA qRT-PCR experimental designs.