Identification of suitable reference genes for hepatic microRNA quantitation

BMC Research Notes

Jatinder K Lamba¹  Weihua Guan²  Yogita Ghodke-Puranik¹  Vishal Lamba¹ 

[1] Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN, USA;Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN, USA

关键词: Endogenous controls;    Normalization;    Real-time quantification;    miRNA expression;    Hepatic;    microRNAs;   

Others  :  1134357
DOI  :  10.1186/1756-0500-7-129

 received in 2013-07-10, accepted in 2014-02-26,  发布年份 2014

【 摘 要 】

Background

MicroRNAs (miRNAs) are short (~22 nt) endogenous RNAs that play important roles in regulating expression of a wide variety of genes involved in different cellular processes. Alterations in microRNA expression patterns have been associated with a number of human diseases. Accurate quantitation of microRNA levels is important for their use as biomarkers and in determining their functions. Real time PCR is the gold standard and the most frequently used technique for miRNA quantitation. Real time PCR data analysis includes normalizing the amplification data to suitable endogenous control/s to ensure that microRNA quantitation is not affected by the variability that is potentially introduced at different experimental steps. U6 (RNU6A) and RNU6B are two commonly used endogenous controls in microRNA quantitation. The present study was designed to investigate inter-individual variability and gender differences in hepatic microRNA expression as well as to identify the best endogenous control/s that could be used for normalization of real-time expression data in liver samples.

Methods

We used Taqman based real time PCR to quantitate hepatic expression levels of 22 microRNAs along with U6 and RNU6B in 50 human livers samples (25 M, 25 F). To identify the best endogenous controls for use in data analysis, we evaluated the amplified candidates for their stability (least variability) in expression using two commonly used software programs: Normfinder and GeNormplus,

Results

Both Normfinder and GeNormplus identified U6 to be among the least stable of all the candidates analyzed, and RNU6B was also not among the top genes in stability. mir-152 and mir-23b were identified to be the two most stable candidates by both Normfinder and GeNormplus in our analysis, and were used as endogenous controls for normalization of hepatic miRNA levels.

Conclusion

Measurements of microRNA stability indicate that U6 and RNU6B are not suitable for use as endogenous controls for normalizing microRNA relative quantitation data in hepatic tissue, and their use can led to possibly erroneous conclusions.

【 授权许可】

   
2014 Lamba et al.; licensee BioMed Central Ltd.

【 图 表 】

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