Email updates

Keep up to date with the latest news and content from BMC Medical Genomics and BioMed Central.

This article is part of the supplement: Proceedings of the 2011 International Conference on Bioinformatics and Computational Biology (BIOCOMP'11)

Open Access Highly Accessed Research

A novel method for the normalization of microRNA RT-PCR data

Rehman Qureshi and Ahmet Sacan*

Author affiliations

Center for integrated Bioinformatics, School of Biomedical Engineering, Science and Health System, Drexel University, 3120 Market Street, Philadelphia, PA 19104, USA

For all author emails, please log on.

Citation and License

BMC Medical Genomics 2013, 6(Suppl 1):S14  doi:10.1186/1755-8794-6-S1-S14

Published: 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.

microRNA; RT-PCR; Normalization; Microarray