Professor Yan Deming, Professor of Shenzhen University, published a new method for circulating microRNA detection

Release date: 2015-10-22

Recently, a research team led by Prof. Yan Deming from the School of Life and Ocean Sciences of Shenzhen University published a recent research report in the Nature Journals of Nature. They used S-Poly(T)Plus real-time PCR to microRNA detection. Improvements.

MicroRNAs (miRNAs) are a class of non-coding small RNAs of approximately 22 nucleotides in length and are widely found in eukaryotes. The main function of miRNA is to inhibit the target mRNA or prevent its translation by binding to the 3' untranslated region of mRNA, thereby inhibiting the expression of genes at the post-transcriptional level, and is widely involved in cell differentiation, proliferation, apoptosis, and individual growth and development. And biological processes such as organ formation. The miRNA in the organism is precisely regulated, and its expression has strict space-time specificity. Studies have shown that miRNA expression abnormalities are closely related to the occurrence of many cancers or other diseases, and miRNAs can exist in the blood in a very stable form. Therefore, circulating miRNAs have great potential as novel biomarkers and can be used for early diagnosis and prognosis evaluation of major diseases such as cancer. However, due to the low level of miRNA in the blood, the sensitivity and specificity of the assay are very high.

Detection techniques based on real-time quantitative PCR (qRT-PCR) have long been considered one of the most sensitive miRNA detection methods. At present, the detection of miRNA by qRT-PCR technology includes the commonly used poly(A) tailing method and Stemloop method. The S-Poly(T) method, originally invented by Prof. Deming's research team, combines the advantages of the traditional Stem-loop method with the Poly (A) tailing method, using ~6 bases that are specifically complementary to the miRNA3' end. 11 Oligod(T)-specific S-Poly(T)? specific reverse transcription primers, at the higher temperature (≥42 °C), the first strand cDNA synthesis of the miRNA after tailing, not only significantly improved S The specificity and thermostability of the complementary binding of the -Poly(T) reverse transcription primer to the miRNA of interest, and the higher reverse transcription efficiency, thereby greatly improving the synthesis of the first strand cDNA of the target miRNA and the subsequent qPCR efficiency. However, in the S-Poly(T) method, Poly(A) tailing and reverse transcription of miRNA are two independent reactions, so there is room for improvement and improvement in terms of ease of operation and reverse transcription efficiency.

The S-Poly(T)Plus technology, recently published in ScientificReports, is a further upgrade of S-Poly(T), which combines the original two-step reaction of tailing and reverse transcription simultaneously, which is not only easy to operate, but also reduces the risk of RNA degradation. The detection time is shortened and the sensitivity of the detection is improved (about 4 times). The researchers further improved the sensitivity of circulating miRNA detection by an improved total RNA isolation method. Through the optimization of various indicators, a total solution for circulating microRNA detection is proposed. 200~300 miRNAs can be detected from 100μl serum or plasma. It is the most sensitive microRNA detection method in the world, especially suitable for finding and Circulating microRNA biomarkers associated with major human diseases.

To validate this improved method, the researchers used this method to quantify miRNA expression profiles in sera from patients with congenital heart disease complicated with pulmonary hypertension, and found two significantly up-regulated miRNAs in the patient population. Five significantly down-regulated miRNAs proved that the method has good application value.

In summary, the simple, sensitive and well-specific circulatory system miRNA detection method developed by the research team of Professor Shen Deming of Shenzhen University provides a new tool for miRNA basic research and human disease diagnosis based on miRNA biomarkers. Further exploration of the mysteries of miRNAs has important implications.

Source: Bio Valley

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