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Method for quantitative correction of mRNA detection in exosome

An exosome, detection value technology, applied in quantitative correction of fluorescent PCR. It can solve the problems of poor stability and inability to distinguish tumor patients from healthy people, and achieve the effect of improving the degree of discrimination, sensitivity and specificity

Active Publication Date: 2021-09-28
3D BIOMEDICINE SCI & TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]The housekeeping gene routinely used for quantitative detection of mRNA by fluorescent PCR is GAPDH. At present, there are many problems mainly in the following aspects: 1. Exosomal GAPDH gene as a housekeeping gene , in the detection results of the same person sampled at different times, the stability is poor
2. The exosomal GAPDH gene, as a housekeeping gene, cannot be well distinguished from tumor patients and healthy people in the detection of exosomal mRNA in different human urine

Method used

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  • Method for quantitative correction of mRNA detection in exosome
  • Method for quantitative correction of mRNA detection in exosome
  • Method for quantitative correction of mRNA detection in exosome

Examples

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Embodiment 1

[0033] Example 1 Screening of urine exosome internal reference genes

[0034] 1.1 Urine exosome preparation and exosome RNA extraction

[0035] 1) Urine sample preparation, the required amount is 30mL. Take 9 urine samples from healthy volunteers, numbered N1-N9; 9 urine samples from urothelial cancer patients, numbered T1-T9;

[0036] 2) Take a urine sample, centrifuge at 3000g at 4°C for 15min, transfer the supernatant to a new centrifuge tube, add 15mL of PEG8000 aqueous solution (concentration 30%), shake and mix, let stand overnight at 4°C, then, 3000g, Centrifuge for 10 min, and use the Qiagen miRNeasy Micro Kit (Qiagen, 217084) to extract exosome RNA from the obtained pellet;

[0037] 1.2 Urine exosomal RNA library construction

[0038] The urine exosome library was constructed using the Ovation SoLo RNA-Seq System kit (NuGen, catalog number: M01406). For the operation method, please refer to the kit instruction manual.

[0039] 1.3 Send the urine exosomal RNA librar...

Embodiment 2

[0049] Example 2 The differential genes screened in urine exosome sequencing samples were corrected using the screened internal reference gene RNF25 / UIMC1

[0050] Through calculation and analysis, we found that for the internal reference gene RNF25, the maximum detection value among the 18 samples was 57.02, and the minimum detection value was 15.31, with a difference of 3.72 times; for UIMC1, the maximum detection value among the 18 samples was 46.36, the minimum detection value The detection value is 13.4, the difference is 3.45 times, and the relative expression of the two genes (RNF25 / UIMC1) trend is between 0.86-1.59 (see Table 3), this fluctuation is relatively small, as shown in Table 3 Show.

[0051] Table 3. Calculation of expression difference and relative expression of internal reference genes

[0052] gene_name N1 N2 N3 N4 N5 N6 N7 N8 N9 RNF25 42.5 57.02 33.15 49 25.57 31.2 35.1 45.76 42.18 UIMC1 36.39 46.36 33.53 42.32 ...

Embodiment 3

[0063] Example 3 qPCR verification of urinary exosome RNF25 gene and UIMC1 gene among different people

[0064] In order to test the relative stability of the RNF25 gene and UIMC1 gene in different samples, we collected 7 urine samples from healthy people, numbered N1-N7, and prepared exosomes and exosomal RNA. The preparation of exosomes and exosomal RNA refers to the process shown in Example 1. After that, the two genes were verified by qPCR. The sequence information of the two primers and probes is shown in Table 7. The qPCR detection uses the Fapon one-step reagent , One step RT-PCR kit (MD027, Faipeng Biotechnology Co., Ltd.), the configuration of the sample well reaction system is as follows:

[0065] Element Volume (μL) RT-PCR Master Mix 10 Enzyme Mix 3 Primer Probe Solution 2 template 5 Enzyme-free water 30

[0066] The reaction process is as follows:

[0067]

[0068] Table 7. RNF25 gene and UIMC1 gene primer and prob...

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Abstract

The invention provides an application of two reference genes UIMC1 and RNF25 in detection of expression level of mRNA in exosome. Moreover, the invention also provides a kit, which comprises a forward primer SEQ ID NO: 1 and a reverse primer SEQ ID NO: 2 of the UIMC1 gene, and a forward primer SEQ ID NO: 4 and a reverse primer SEQ ID NO: 5 of the RNF25 gene. The expression of the two reference genes is relatively stable at different time points of the same individual and among different individuals, especially the relative expression level between the two genes is more stable, and the reference genes can be used for correcting the expression level of mRNA in exosomes and can also be used for detecting cancers of the urinary tract system.

Description

technical field [0001] The present invention is to provide a pair of internal reference genes for exosome mRNA detection, which are used for quantitative correction of fluorescent PCR. Background technique [0002] There are different gene expression patterns in different tissues in the human body, but some gene products are necessary to maintain basic cell functions and are stably expressed in all cells. These genes are called housekeeping genes (HK genes) 1 . HK genes can often be used to calibrate gene expression measurements 2 . GAPDH is reported to be a more commonly used housekeeping gene in cells and tissues 3 . [0003] Exosome, also known as exosome, with a diameter of 30-150nm, is a small vesicle wrapped by a lipid bimolecular membrane released into the extracellular environment after the fusion of intracellular multivesicular body (MVB) and cell membrane. Vessels, including nucleic acids (DNA, mRNA, microRNA (miRNA), lncRNA, circRNA, etc.), proteins 4 . Uri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6886C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 钱祺王晓楠郭由兵汤华军张亚楠
Owner 3D BIOMEDICINE SCI & TECH CO LTD
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