Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis.

Samira Mohammadi-Yeganeh (1) , Mahdi Paryan (1) , Siamak Mirab Samiee (2) , Masoud Soleimani (3, 4) , Ehsan Arefian (4) , Keyhan Azadmanesh (5) , Ehsan Mostafavi (6) , Reza Mahdian (1) , Morteza Karimipoor (1)

1 Biotechnology Research Center
2 Food and Drug Laboratory Research Center
3 Department of Hematology
4 Department of Molecular Biology and Genetic Engineering
5 Institut Pasteur d'Iran
6 Department of Epidemiology

Development of a rapid and accurate quantification method for the detection of microRNAs (miRNAs) has been desired, in particular, when they are differently expressed in normal and pathological conditions. However, various methods for the quantification of small non-coding RNAs as well as miRNAs have been described. These methods mainly include hybridization-based approaches such as primer extension, northern blotting, microarray profiling, and reverse transcription (RT) PCR. Here, we developed a simple and rapid method based on stem-loop primer-based real-time PCR assay for sensitive and accurate detection of mature miRNAs. Initially, a miRNA-specific stem-loop RT primer is used for RT, which is followed by TaqMan real-time PCR assay using specific forward primer in combination with universal reverse primer and TaqMan probe. The assay has shown high sensitivity (≤50 copies/reaction) for miRNA detection in two breast cancer cell lines, MCF-7 and MDA-MB-231. This assay might be implicated as a rapid and cost effective method for the detection of small non-coding RNAs.

Mots clés: miRNA; Stem-loop RT-PCR; TaqMan real-time PCR

Domaines: Génétique

Samira Mohammadi-Yeganeh, Mahdi Paryan, Siamak Mirab Samiee, Masoud Soleimani, Ehsan Arefian, et al.. Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis.. Molecular Biology Reports, 2013, 40 (5), pp.3665-74. ⟨10.1007/s11033-012-2442-x⟩. ⟨pasteur-00821223⟩