This article is part of the supplement: Proceedings of the 2011 International Conference on Bioinformatics and Computational Biology (BIOCOMP'11)
A novel method for the normalization of microRNA RT-PCR data
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BMC Medical Genomics 2013, 6(Suppl 1):S14 doi:10.1186/1755-8794-6-S1-S14Published: 23 January 2013
MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate mRNA transcript levels and translation. Deregulation of microRNAs is indicated in a number of diseases and microRNAs are seen as a promising target for biomarker identification and drug development. miRNA expression is commonly measured by microarray or real-time polymerase chain reaction (RT-PCR). The findings of RT-PCR data are highly dependent on the normalization techniques used during preprocessing of the Cycle Threshold readings from RT-PCR. Some of the commonly used endogenous controls themselves have been discovered to be differentially expressed in various conditions such as cancer, making them inappropriate internal controls.
We demonstrate that RT-PCR data contains a systematic bias resulting in large variations in the Cycle Threshold (CT) values of the low-abundant miRNA samples. We propose a new data normalization method that considers all available microRNAs as endogenous controls. A weighted normalization approach is utilized to allow contribution from all microRNAs, weighted by their empirical stability.
The systematic bias in RT-PCR data is illustrated on a microRNA dataset obtained from primary cutaneous melanocytic neoplasms. We show that through a single control parameter, this method is able to emulate other commonly used normalization methods and thus provides a more general approach. We explore the consistency of RT-PCR expression data with microarray expression by utilizing a dataset where both RT-PCR and microarray profiling data is available for the same miRNA samples.
A weighted normalization method allows the contribution of all of the miRNAs, whether they are highly abundant or have low expression levels. Our findings further suggest that the normalization of a particular miRNA should rely on only miRNAs that have comparable expression levels.