阅读说明：本技术 一种环状rna的检测方法及其在成体神经干细胞鉴定中的应用 (Detection method of circular RNA and application of circular RNA in adult neural stem cell identification ) 是由 王雪 谢方 钱令嘉 赵云 宋子甜 于 2019-11-21 设计创作，主要内容包括：一种环状RNA的检测方法及其在成体神经干细胞鉴定中的应用。本发明提供一种circ-NSC基因、该基因制备鉴定成体干细胞的试剂中的应用及检测该基因的试剂盒,其中,所述基因的cDNA序列如SEQ ID NO:1所示；所述试剂盒包括：DNA聚合酶、缓冲液、去离子水、扩增引物对,所述扩增引物对核苷酸序列如SEQ ID NO:2和SEQ ID NO:3所示。本发明通过高通量RNA测序技术,鉴定出在SVZ区表达的circRNAs,获得circRNAs的全长序列,通过功能预测发现circ-NSC序列上存在2个miR-138-5p的可能结合位点,提示该circRNA可能通过竞争性结合miR-138-5p发挥NSC调控作用,根据circ-NSC的序列设计了特异性扩增引物对,通过PCR实验验证了circ-NSC在SVZ中特异性表达,提示其在NSC的鉴定中具有一定应用价值。(A method for detecting circular RNA and application thereof in adult neural stem cell identification. The invention provides a circ-NSC gene, application of the gene in preparing a reagent for identifying adult stem cells and a kit for detecting the gene, wherein the cDNA sequence of the gene is shown as SEQ ID NO. 1; the kit comprises: DNA polymerase, buffer solution, deionized water and an amplification primer pair, wherein the nucleotide sequence of the amplification primer pair is shown as SEQ ID NO. 2 and SEQ ID NO. 3. According to the invention, the circRNAs expressed in the SVZ region are identified by a high-throughput RNA sequencing technology, the full-length sequence of the circRNAs is obtained, 2 possible binding sites of miR-138-5p exist on the circ-NSC sequence through function prediction, the circRNA is prompted to play an NSC regulation function by competitively binding miR-138-5p, a specific amplification primer pair is designed according to the sequence of the circ-NSC, the specific expression of the circ-NSC in the SVZ is verified through a PCR experiment, and the circ-NSC is prompted to have a certain application value in the identification of the NSC.)

1. A circ-NSC gene is characterized in that the cDNA sequence of the gene is shown as SEQ ID NO. 1.

2. Use of the circ-NSC gene of claim 1 for the preparation of a reagent for the identification of adult stem cells.

3. A kit for detecting the circ-NSC gene of claim 1, comprising: DNA polymerase, buffer solution, deionized water and an amplification primer pair, and is characterized in that the nucleotide sequence of the amplification primer pair is shown as SEQ ID NO. 2 and SEQ ID NO. 3.

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a detection method of circular RNA and application of the detection method in adult neural stem cell identification.

Background

Neural Stem Cells (NSCs) refer to a very small population of cells with long-term self-renewal and multipotentiality. In 1992, Reynolds and Richards, in turn, isolated neural stem cells from the striatum and hippocampus of adult mice and demonstrated that they could differentiate into neurons, astrocytes and oligodendrocytes. Subsequent studies have found that neural stem cells are present predominantly in the subventricular zone (SVZ) and the hippocampal dentate zone (SGZ) of the adult mammalian brain. Domestic and foreign studies show that after brain injury, NSCs in regions such as SVZ and the like can be activated, proliferated and migrated to the injury site, and differentiated into corresponding nerve cells to realize structural and functional compensation. In recent years, NSC transplantation has attracted more and more attention in the treatment of nervous system diseases, i.e., NSCs are formed by reprogramming somatic cells of a patient in vitro and then transplanted into the brain of the patient, so as to achieve the purpose of treatment. At present, a plurality of animals and preclinical experiments probe the application value of NSC transplantation in nervous system diseases such as Parkinson's disease, Alzheimer's disease, cerebral arterial thrombosis and the like. Therefore, it is important to identify NSCs accurately and efficiently. NSC has no obvious difference in morphology with other cells, and is currently mainly identified by biomarkers, such as CD133, Nestin, Sox2 and the like. Although these molecules are highly expressed in NSCs, they are also expressed in other neural cells or other tissue stem cells, and thus are less specific. It was therefore found that novel NSC-specific markers have a critical role in the identification of NSCs.

Circular RNA (circular RNA) is a special non-coding RNA, and a single-stranded RNA molecule forms a circular structure through covalent bonding and does not have a 5 'terminal cap and a 3' terminal polyA tail structure. The circRNA is widely existed in eukaryotic cells, and the expression level of part of the circRNA is even more than 10 times higher than that of mRNA. The circular structure of circRNA makes it less susceptible to degradation by exonucleases and more stable than linear RNA. In addition, circRNA is also highly conserved and tissue-specific. Initially, circRNA was considered a by-product of the mis-or splicing process and had no biological function. However, recent studies show that circRNA can play an important role in regulation at the level of transcription or after transcription by binding miRNA or protein, and participate in various physiopathological processes. Research shows that the circRNA plays an important role in the occurrence and development of nervous system diseases. Furthermore, we previously found by RNA sequencing that there was a circRNA specifically expressed in NSCs. The existing biomarkers CD133, Nestin, Sox2 and the like commonly used for identifying NSC have the defect of weak specificity, and compared with the circRNA which has the characteristics of high expression, high conservation, high stability, high tissue specificity and the like, the circRNA can be an ideal biomarker for identifying NSC.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide a circ-NSC gene as a biomarker for identifying adult neural stem cells.

The invention firstly provides a circ-NSC gene which is characterized in that the cDNA sequence of the gene is shown as SEQ ID NO. 1.

The invention also provides application of the circ-NSC gene in preparing a reagent for identifying adult stem cells.

In addition, the present invention also provides a kit for detecting the circ-NSC gene, the kit comprising: DNA polymerase, buffer solution, deionized water and an amplification primer pair, wherein the nucleotide sequence of the amplification primer pair is shown as SEQ ID NO. 2 and SEQ ID NO. 3.

In the present invention, we identified circRNAs expressed in SVZ region by high throughput RNA sequencing technology to obtain full length sequences of the circRNAs. Through functional prediction, 2 possible binding sites of miR-138-5p exist on the sequence of the rat _ circ, chr15, 9915223 and 9915671, which indicates that the circRNA can play NSC regulation role by competitively binding to the miR-138-5p and is named as circ-NSC. A specific amplification primer pair is designed according to the sequence of the circ-NSC, and the specific expression of the circ-NSC in SVZ is verified through a PCR experiment, which indicates that the primer pair has a certain application value in the identification of the NSC.

Drawings

FIG. 1: the expression level of SVZ-specific circRNA in SVZ (P < 0.05);

FIG. 2: the expression level of circ-NSCs in different brain regions in RNA sequencing results;

FIG. 3: PCR detects the expression of circ-NSC in different brain regions.

Detailed Description

The embodiments are described in detail below with reference to the accompanying drawings.