Identification of most stable endogenous control genes for microRNA quantification in the developing mouse lung.

Wafae Bouhaddioui,Yves Tremblay,Pierre R Provost,Baochuan Lin

doi:10.1371/journal.pone.0111855

Abstract

MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies.

Highlights

MicroRNAs are small non coding RNAs of,22 nucleotides [1]
The lung miRNA expression profile is highly conserved among mammalian species [8,9]. miRNAs are involved in homeostasis and lung development [10], inflammation and viral infections [11,12], and many pulmonary diseases such as cancer [13] and chronic obstructive pulmonary disease [14]
Because the developing lung is changing over time, quantification of miRNA expression requires careful selection of endogenous control genes according to the studied period of development

Summary

Introduction

MicroRNAs (miRNAs) are small non coding RNAs of ,22 nucleotides [1]. They are endogenous regulatory molecules that negatively regulates gene expression [1]. The two first discovered miRNAs were reported in 1993 (Lin 4) and 2000 (let-7) [2,3]. This class of molecules are involved in different physiological and pathological processes. It was shown that over 60% of human protein-coding genes are conserved targets of miRNAs [7]. The lung miRNA expression profile is highly conserved among mammalian species [8,9]. Knowledge on the role of miRNAs in lung development is still limited and based mainly on data from animal models

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Results

Conclusion