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Application of miRNA-30a in preparation of diagnostic reagents as marker molecules

A technology of 1.mirna-30a and 5.mirna-30a, which is applied in the field of biomedicine and can solve the problems of variable factors, poor stability and specificity, etc.

Inactive Publication Date: 2015-01-28
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The purpose of the present invention is to provide a use of miRNA-30a as a marker molecule in the preparation of diagnostic reagents, which solves the problem of the stability and specificity of molecular markers in the prior art for diagnosing acute kidney injury induced by contrast agents. Less flexible, technical problems susceptible to many pre-existing variables

Method used

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  • Application of miRNA-30a in preparation of diagnostic reagents as marker molecules
  • Application of miRNA-30a in preparation of diagnostic reagents as marker molecules
  • Application of miRNA-30a in preparation of diagnostic reagents as marker molecules

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Chip screening of target miRNA specifically expressed in human kidney

[0041] 1. Construction of CI-AKI rat model

[0042] The present invention establishes a CI-AKI rat model by injecting a hypotonic contrast agent through the tail vein on the basis of water deprivation for 3 days and diuretic "pretreatment". This is a new type of CI-AKI rat model established by the inventors, and the specific protocol can be found in relevant literature (Sun SQ, et al. Int Journal of Cardiol 172(2014) e48-50).

[0043] 2. Differential expression profile analysis of miRNA microarray

[0044] 1) miRNA chip preparation

[0045] On the basis of successful modeling using the above method, plasma and kidneys of CI-AKI rats were collected 8 hours after modeling for sample loading on the chip. Using Agilent miRNAs 10.0version chip technology, plasma and kidney total RNA were used for loading, and phosphorylated Cyanine3-pCp was used for labeling, followed by chip hybridization, ...

Embodiment 2

[0053] Example 2 Verification of the specific expression of the target miRNA kidney

[0054] The total RNA of rat tissues was extracted by the classic Trizol method, and real-time PCR was used to detect the expression levels of target miRNAs in rat tissues. At the same time, the expression levels of human tissues were compared by searching published literature on the expression profile of miRNAs in human tissues. figure 2 It is the result of the expression profile of miRNA-30a in each tissue of rat (A) and human (B). The expression of miRNA-30a in the kidney of rat and human is 2 times or more than that of other organs, so it is considered that the miRNA kidney specificity is high.

Embodiment 3

[0055] Example 3 Extraction of plasma microRNA

[0056] The present invention adopts the two-step centrifugation method to blood plasma, has removed blood cell and cell debris in blood, and RNA extraction adopts mirVana PARIS Kit (Applied Biosystem, P / N AM1556), and this is a kind of RNA that is applicable to blood plasma or serum. Reagent test kit. At present, there is no recognized internal reference for RNA quantification in plasma. In the present invention, an exogenous internal reference cel-miR-39 (miRNA expressed by nematodes, which is not expressed in the human body) is added to the RNA extraction process to correct RT-PCR detection results of target miRNAs.

[0057] Plasma sample pretreatment method: collect whole blood in EDTA anticoagulant tubes (working concentration: 1.8mg / ml), first centrifuge at 4°C, 820g for 10min to remove blood cells, and then centrifuge the supernatant at 4°C, 16000g for 10min to remove cells Finally, pipette the supernatant into a 1.5ml R...

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Abstract

The invention provides application of miRNA-30a in preparation of reagents for diagnosing contrast-induced acute kidney injury as marker molecules. The invention further provides application of miRNA-30a combined with any one or two of miRNA-30e and miRNA-188 in preparation of reagents for diagnosing contrast-induced acute kidney injury as marker molecules. The invention further provides a kit containing miRNA-30a. Experiments prove that miRNA-30a can be used for early diagnosis of CI-AKI (contrast-induced acute kidney injury). When miRNA-30a is used alone for diagnosing CI-AKI, the sensitivity is 56.3% and the specificity is 94.4%; and when miRNA-30a is combined with miRNA-30e and miRNA-188 for diagnosing CI-AKI, the sensitivity is 71.8% and the specificity is 88.7%. The adoption of the marker in early diagnosis of CI-AKI has great application prospects and market prospects.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a molecular marker, specifically the use of miRNA-30a as a marker molecule in the preparation of diagnostic reagents. Background technique [0002] Contrast Induced Acute Kidney Injury (CI-AKI) refers to new acute renal injury or aggravation of renal insufficiency after contrast agent administration, excluding other factors. CI-AKI is the third cause of iatrogenic acute renal failure, accounting for 11% of iatrogenic acute renal failure, and it is mainly seen clinically in patients undergoing coronary interventional surgery. In recent years, with the increase of the incidence of coronary heart disease in my country and the extensive development of coronary intervention technology, the incidence of CI-AKI and the absolute number of cases have been increasing year by year. [0003] Although most CI-AKI patients show a transient increase in serum creatinine level, and the pro...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/156C12Q2600/178
Inventor 何奔沈玲红张拓孙士群
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE