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


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.


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





Phosphatase and tensin homolog


Phosphoinositide 3-kinases


Interleukin 6


Tumor necrosis factor alpha


Interleukin 1 beta


Phosphate-buffered saline



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.


  1. 1.
    Kopelman PG (2000) Obesity as a medical problem. Nature 404(6778):43–635CrossRefGoogle Scholar
  2. 2.
    Guay C, Regazzi R (2013) Circulating microRNAs as novel biomarkers for diabetes mellitus. Nat Rev Endocrinol 9(9):513–550CrossRefGoogle Scholar
  3. 3.
    Tothill IE (2009) Biosensors for cancer markers diagnosis. Semin Cell Dev Biol 20:55–62CrossRefGoogle Scholar
  4. 4.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297CrossRefGoogle Scholar
  5. 5.
    Buchanan TA, Xiang AH (2005) Gestational diabetes mellitus. J Clin Investig 115(3):485–491CrossRefGoogle Scholar
  6. 6.
    Guay C, Roggli E, Nesca V et al (2011) Diabetes mellitus, a microRNA-related disease? Transl Res 157(4):253–264CrossRefGoogle Scholar
  7. 7.
    de Planell-Saguer M, Rodicio MC (2013) Detection methods for microRNAs in clinic practice. Clin Biochem 46(10–11):869–878CrossRefGoogle Scholar
  8. 8.
    Zampetaki A et al (2010) Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circ Res 107:810–817CrossRefGoogle Scholar
  9. 9.
    Sekar D, Islam VIH, Thirugnanasambantham K et al (2014) Relevance of miR-21 in HIV and non-HIV-related lymphomas. Tumour Biol 35(9):8387–8393CrossRefGoogle Scholar
  10. 10.
    Dey N, Das F, Mariappan MM et al (2011) microRNA-21 orchestrates high glucose-induced signals to TORC1 for renal cell pathology in diabetes. J Biol Chem 286(29):68–75CrossRefGoogle Scholar
  11. 11.
    Roy S, Khanna S, Hussain SRA (2009) MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue. Cardiovasc Res 82(1):21–29CrossRefGoogle Scholar
  12. 12.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res 29(9):e45CrossRefGoogle Scholar
  13. 13.
    Olivieri F, Rippo MR, Prattichizzo F et al (2013) Toll like receptor signaling in “inflammaging”: microRNA as new players. Immun Ageing 10(1):57–68CrossRefGoogle Scholar
  14. 14.
    Li S, Chen X, Zhang H et al (2009) Differential expression of microRNAs in mouse liver under aberrant energy metabolic status. J Lipid Res 50(9):1756–1765CrossRefGoogle Scholar
  15. 15.
    Meng S, Cao JT, Zhang B, Wang CQ et al (2012) Down regulation of microRNA-126 in endothelial progenitor cells from diabetes patients, impairs their functional properties, via target gene Spred-1. J Mol Cell Cardiol 53(1):64–72CrossRefGoogle Scholar

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

Personalised recommendations