Reference gene for detecting expression level of nucleic acid in exosome and application thereof

An internal reference gene and expression level technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of poor data repeatability and instability, and improve the amplification effect and detection efficiency , high accuracy and high stability

Active Publication Date: 2019-10-18
宽盈医疗科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low content of nucleic acid in exosomes, when the expression of target genes in exosomes is detected by fluorescent PCR, the data repeatability is often poor. One of the important reasons is that in the current research on exosome nucleic acid, there is no Stable internal control to calibrate the expression of the target gene

Method used

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  • Reference gene for detecting expression level of nucleic acid in exosome and application thereof
  • Reference gene for detecting expression level of nucleic acid in exosome and application thereof
  • Reference gene for detecting expression level of nucleic acid in exosome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Through the following experiments, the inventors optimized the different exons of the gene according to the CT value stability of the different exons of the internal reference gene, and obtained the preferred exons of each internal reference gene.

[0051] Design primers for each gene exon: ACTB covers exons 1, 2, 3, 4, 5, and 6; EEF2 covers exons 1, 2, 12, 13, 14, and 15; MSN covers exons 1, 2 , 3, 4, 12 and 13 exons; TLN1 covers exons 1, 2, 3, 27, 56 and 57.

[0052] Collect blood samples from 15 clinically diagnosed patients with different diseases, and process the incoming samples as follows: use an exosome extraction kit (QIAGEN: exoEasy Maxi Kit) to extract the total exosomes in the sample serum come out. Subsequently, the extracted exosomes were extracted with a ribonucleic acid extraction kit (QIAGEN company: miRNeasy MicroKit) to extract all the ribonucleic acids in the exosomes. The extracted ribonucleic acid was used reverse transcription kit (Takara compan...

Embodiment 2

[0056] In order to further optimize the formula model of the internal reference gene combination, the inventors conducted the following research experiments to compare the differences of the internal reference model when different exon combinations were selected. Among them, the internal reference model is CONCT=0.7×ACTB CT +0.1×EEF2 CT +0.1×MSN CT +0.1×TLN1 CT .

[0057] Combination 1: ACTB selects exon 6, EEF2 selects exon 15, MSN selects exon 4, and TLN1 selects exon 57.

[0058] Combination 2: ACTB selects exon 5, EEF2 selects exon 12, MSN selects exon 12, and TLN1 selects exon 27.

[0059] Combination 3: ACTB selects exon 4, EEF2 selects exon 1, MSN selects exon 1, and TLN1 selects exon 1.

[0060] Using the blood samples of 10 clinically diagnosed patients with different diseases, compare the experimental results of the internal reference model under the above three combinations (combination 1, combination 2 and combination 3), such as Image 6 shown. For specific...

Embodiment 3

[0064] The inventors compared the expression level of the target gene in exosomes by adding an exogenous reference gene (a synthetic GH1 (growth hormone 1) gene mRNA whose sequence is shown in SEQ ID NO: 22) and the CONCT model (combination 1) strengths and weaknesses in the analysis.

[0065] The PTEN gene with high expression in prostate cancer cells was selected as the verification target. The recognized GAPDH was selected as the internal reference gene for cellular RNA, and the CONCT model and exogenous spike-in were used as internal references for the detection of PTEN expression in exosomes.

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Abstract

The invention discloses an application of a reference gene in preparation of a reagent or a kit for detecting expression level of a target gene in an exosome, and a reference gene combination and an application of the reference gene combination in preparation of a reagent for detecting expression level of a target gene in an exosome. The invention also provides a reagent combination and a method for detecting expression level of a target gene in an exosome. Compared to an exogenous reference gene, the reference gene of the present invention can achieve higher accuracy of detection results.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to internal reference genes and their applications. More specifically, it relates to an internal reference gene for detecting the expression level of nucleic acid in exosomes and its application. Background technique [0002] Gene expression has a wide range of applications in the life sciences. The detection of gene expression is of great significance to the analysis of gene function and the search for genes related to specific phenotypes and diseases. Finding the factors that affect the expression of functional genes has an important guiding role in analyzing the causes of related diseases and finding suitable treatment methods. Gene expression detection methods widely used include real-time fluorescent quantitative PCR (Quantitative real-time RT-PCR, qRT-PCR), Northern blotting (Northern blotting), ribonuclease protection assay (ribonuclease protection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686
Inventor 韩君
Owner 宽盈医疗科技(上海)有限公司
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