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.
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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
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Acknowledgements
This work was financially supported by graduate study of University of Kashan under Grant No. 572212/04.
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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.
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Andoorfar, S., Hosseini Tafreshi, S.A. & Rezvani, Z. Assessment of the expression level of miRNA molecules using a semi-quantitative RT-PCR approach. Mol Biol Rep 46, 5057–5062 (2019). https://doi.org/10.1007/s11033-019-04959-5
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DOI: https://doi.org/10.1007/s11033-019-04959-5