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Assessment of the expression level of miRNA molecules using a semi-quantitative RT-PCR approach

  • Shima Andoorfar
  • Seyed Ali Hosseini TafreshiEmail author
  • Zahra Rezvani
Original Article

Abstract

Type 2 diabetes is one of the most prevalent diseases, which increases resistance to insulin in target tissues. The measurement of miRNAs quantity is a molecular approach for diagnosis of diabetes. miRNAs are small non-coding RNA strings of 21–23 long nucleotides that act as inhibitors in proteins translation. Several methods including Northern blot, qRT-PCR and Microarray have been used for diagnosis of miRNA molecules. Real time PCR is an expensive and accurate quantitative method that is widely used in miRNA studies. The miR-21 is an important miRNA in diabetes. In this study, for the first time, a semi-quantitative protocol was developed to quantify different amounts of a synthetic miR-21. In addition to semi-quantitative method, the miR-21 quantity was determined by quantitative method in several patients with type 2 diabetes and healthy people. The results indicated that there was a direct relationship between the amount of synthetic miR-21 and the intensity of the PCR bands. We also showed that the expression of miR-21 in people with type 2 diabetes increased compared to healthy people. The results were observed by both quantitative and semi-quantitative methods. The real-time RT-PCR was more sensitive than semi-quantitative PCR in identification of miRNAs. However, semi-quantitative PCR method benefited from higher simplicity and lower costs for defining general patterns of miRNA expression.

Keywords

Type 2 diabetes miR-21 Semi-quantitative PCR Real-time RT-PCR 

Abbreviations

miRNA

MicroRNA

PTEN

Phosphatase and tensin homolog

PI3K

Phosphoinositide 3-kinases

IL-6

Interleukin 6

TNF-α

Tumor necrosis factor alpha

IL-1B

Interleukin 1 beta

PBS

Phosphate-buffered saline

Notes

Acknowledgements

This work was financially supported by graduate study of University of Kashan under Grant No. 572212/04.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was granted by the Aklaghezisti Ethical Committee at University of Kashan. All procedures were in accordance with the approved ethical standards of the Ethical Committee.

Informed consent

Written informed consent obtained from all healthy and diabetic people.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Shima Andoorfar
    • 1
  • Seyed Ali Hosseini Tafreshi
    • 1
    Email author
  • Zahra Rezvani
    • 1
  1. 1.Biotechnology Division, Department of Cell and Molecular Biology, Faculty of ChemistryUniversity of KashanKashanIran

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