Sains Malaysiana 44(10)(2015): 1481–1488

U6 snRNA is a Suitable Endogenous Control for microRNA-124 and -134 in Cultured Rat Hippocampal Neurons

(U6 snRNA adalah kawalan Endogen yang sesuai untuk microRNA-124 dan -134 dalam neuron hipokampus tikus berkultur)

AI SZE CHING & AZLINA AHMAD-ANNUAR*

Department of Biomedical Science, Faculty of Medicine, University of Malaya,

50603 Kuala Lumpur, Malaysia

Received: 21 July 2014/Accepted: 23 June 2015

ABSTRACT

As researchers seek to determine the cellular mechanisms underlying biological processes, they have turned to analyze the functional role of microRNAs to understand this process in details. Here, we investigated the expression pattern of two microRNAs, miR-124 and -134 in maturing neurons and found that the choice of endogenous controls influenced the observed expression levels of these microRNAs. We have cultured rat hippocampal neurons and performed quantitative PCR on the microRNAs using Taqman gene expression assays. The expression of miRNAs was normalised with selected endogenous controls. Using BestKeeper and NormFinder software, we found that 18S rRNA and 5S rRNA to be unsuitable as endogenous controls in this system, while normalising to U6 snRNA produced more consistent results. Our study would like to highlight the importance of empirically testing proposed endogenous controls in any model system before data interpretation is carried out.

Keywords: Endogenous controls; hippocampal neuron culture; microRNAs; miR-124; miR-134

ABSTRAK

Dalam usaha untuk menentukan mekanisme sel dalam proses biologi, para penyelidik telah beralih untuk menganalisis peranan fungsi mikroRNA untuk memahami proses ini dengan lebih mendalam. Kami telah menentukan tahap ekspresi untuk dua jenis mikroRNA, miR-124 dan -134 dalam sel-sel neuron yang semakin matang dan mendapati bahawa pilihan untuk gen endogen boleh mempengaruhi tahap ekspresi yang diperhatikan untuk mikroRNA tersebut. Kami telah mengkultur sel neuron daripada hipokampus tikus dan menggunakan teknik PCR kuantitatif dengan asai ekspresi gen Taqman. Ekspresi miRNA gen telah dipenormal dengan gen endogen. Dengan menggunakan perisian BestKeeper dan NormFinder, kami mendapati bahawa rRNA 18S dan rRNA 5S merupakan gen endogen yang tidak sesuai dalam sistem ini, manakala keputusan lebih konsisten apabila snRNA U6 digunakan. Hasil penyelidikan kami menunjukkan kepentingan untuk menggunakan pendekatan secara empirik semasa menimbangkan pilihan gen endogen yang ingin digunakan.

Kata kunci: Kawalan endogen; kultur neuron hipokampus; mikroRNAs; miR-124; miR-134

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*Corresponding author; email: azlina_aa@um.edu.my