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Use of miRNA-188 as a marker molecule in the preparation of diagnostic reagents

A miRNA-188, 1.miRNA-188 technology, applied in the field of biomedicine, can solve the problems of variable factors, poor stability and specificity, etc.

Inactive Publication Date: 2016-02-10
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • 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-188 as a marker molecule in the preparation of diagnostic reagents, which solves the problem of the stability and specificity of molecular markers in the diagnosis of acute kidney injury induced by contrast agents in the prior art. Less flexible, technical problems susceptible to many pre-existing variables

Method used

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  • Use of miRNA-188 as a marker molecule in the preparation of diagnostic reagents
  • Use of miRNA-188 as a marker molecule in the preparation of diagnostic reagents
  • Use of miRNA-188 as a marker molecule in the preparation of diagnostic reagents

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Chip screening target miRNA specifically expressed in human kidney

[0040] 1. Construction of CI-AKI rat model

[0041] 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 present inventors. For details, please refer to relevant literature (SunSQ, et al. Int Journal of Cardiol 172 (2014) e48-50).

[0042] 2. Differential expression profile analysis of miRNA microarray

[0043] 1) miRNA chip preparation

[0044] 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 AgilentmiRNAs10.0version chip technology, plasma and kidney total RNA were used for loading, and phosphorylated Cyanine3-pCp was used for labeling, followed by chip hybridization, washing an...

Embodiment 2

[0052] Example 2 Verifies the specific expression of target miRNA kidney

[0053] The total RNA of rat tissues was extracted by the classic Trizol method, and the expression levels of target miRNAs in rat tissues were detected by real-time PCR. At the same time, the expression levels of human tissues were compared by searching published literature on miRNAs expression profiles in human tissues. figure 2 It is the result of the expression profile of miR-188 in each tissue of rat (A) and human (B). The expression of miR-188 in the kidney of rats and humans is 2 times or more than that of other organs, so it is considered that the miRNA kidney specificity is high.

Embodiment 3

[0054] Example 3 Extraction of plasma microRNA

[0055] The present invention adopts the two-step centrifugation method to blood plasma, has removed blood cell and cell fragment in blood, and RNA extraction adopts mirVanaPARISKit (Applied Biosystem, P / NAM1556), and this is a kind of test kit applicable to RNA in blood plasma or serum. 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.

[0056] 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 RNase / DNase-free ...

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Abstract

The invention provides applications of miRNA-188 in preparation of a contrast-induced acute kidney injury diagnosis reagent as a labelled molecule. The invention further provides applications of miRNA-188 combined with any one or two of miRNA-30a and miRNA-e as labelled molecules to prepare a contrast-induced acute kidney injury diagnosis reagent. The invention further provides a kit containing miRNA-188. Experiments verify that the miRNA-188 can be used for early diagnosis of CI-AKI. The sensitivity and specificity of the miRNA-188 for diagnosing CI-AKI are respectively 52.11% and 92.96%; and the sensitivity and specificity of the miRNA-188 combined with the miR-30a and miR-e for diagnosing CI-AKI are respectively 71.8% and 88.7%. The marker for early diagnosis of CI-AKI has tremendous 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-188 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/178
Inventor 何奔沈玲红张拓孙士群
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE