Exp Mol Med.  2011 Jun;43(6):358-366. 10.3858/emm.2011.43.6.039.

Identification of miR-23a as a novel microRNA normalizer for relative quantification in human uterine cervical tissues

• Shen Y
• Li Y
• Ye F
• Wang F
• Wan X
• Lu W
• Xie X

• KMID: 1107377
• DOI: http://doi.org/10.3858/emm.2011.43.6.039

Abstract

Quantitative real-time RT-PCR (RT-qPCR) is being widely used in microRNA expression research. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in microRNA RT-qPCR studies. The aim of this study was to identify the most stable reference gene(s) for quantification of microRNA expression analysis in uterine cervical tissues. A microarray was performed on 6 pairs of uterine cervical tissues to identify the candidate reference genes. The stability of candidate reference genes was assessed by RT-qPCR in 23 pairs of uterine cervical tissues. The identified most stable reference genes were further validated in other cohort of 108 clinical uterine cervical samples: (HR-HPV- normal, n = 21; HR-HPV+ normal, n = 19; cervical intraepithelial neoplasia [CIN], n = 47; cancer, n = 21), and the effects of normalizers on the relative quantity of target miR-424 were assessed. In the array experiment, miR-26a, miR-23a, miR-200c, let-7a, and miR-1979 were identified as candidate reference genes for subsequent validation. MiR-23a was identified as the most reliable reference gene followed by miR-191. The use of miR-23a and miR-191 to normalize expression data enabled detection of a significant deregulation of miR-424 between normal, CIN and cancer tissue. Our results suggested that miR-23a and miR-191 are the optimal reference microRNAs that can be used for normalization in profiling studies of cervical tissues; miR-23a is a novel microRNA normalizer.

Keyword

gene expression profiling; microRNAs; real-time polymerase chain reaction; uterine cervical neoplasms

MeSH Terms

Cervical Intraepithelial Neoplasia/diagnosis/genetics/*metabolism/pathology
Cervix Uteri/*metabolism/pathology
Early Detection of Cancer
Female
Gene Expression Profiling/*standards
Humans
MicroRNAs/genetics/*metabolism/standards
Microarray Analysis
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction
Uterine Cervical Neoplasms/diagnosis/genetics/*metabolism/pathology