Normalizing to the geometric mean of all detected miRs in a large screen provides an effective benchmark; however, the need for costly high-throughput assays limits the practical use of this strategy for large numbers of samples. Therefore, an optimal alternative would be the identification of one or a few specific internal reference controls that can replicate the results of MCR normalization. In the present study, we leveraged the results of a previous global screen of plasma miRNAs in samples from PAH patients, in order to facilitate the de novo identification of circulating miRNAs that could function as internal reference controls. From among an initial pool of over 200 candidate miRNAs that were detectable in all subjects, miR-142-3p and miR-320a were selected by the NormFinder algorithm as the most stable combination. However, based on the observation that a number of miRs exhibited similar levels of stability in the Normfinder analysis, it is possible that other circulating miRNAs may also be able to serve as GW7647 useful reference controls, such as miR-222. Of note, the geNorm algorithm selected a different pair of miRNAs as the most stable combination, though these candidates did not perform as well as miR-142-3p and miR-320a in subsequent evaluations. Unlike geNorm, the NormFinder algorithm not only measures the overall variation across subjects, but also examines the system aticvariation between groups of related subjects, and therefore may improve the selection of reference controls that are less likely to be affected by the experimental condition under investigation. The NormFinder algorithm may also offer a more robust measure of gene expression stability, because itis less susceptible to the effects of co-regulated genes. In contrast, geNorm relies on a pair-wise comparison approach to rank the expression stability of genes based on the similarity of their expression profiles, so coregulated genes maybe ranked highly because of their tendency to show similar expression profiles, independent of their actual expression stability. The quality of the internal reference controls was judged by several TDZD-8 criteria.
The identification of internal reference controls can replicate the results of MCR normalization
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