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Serum/plasma miRNA composition and use thereof

A technology of composition and primer composition, applied in the field of molecular biology, can solve problems such as incomplete clarification of the relationship, and achieve the effects of quickly and accurately grasping the patient's condition, being easy to obtain, and improving accuracy

Inactive Publication Date: 2011-04-20
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes different types of microRNAs (microribonucleic acids) found within blood cells called HepG2 cytoskeleton DNA (Human Glycoprotein 2). These tiny nonproteins play crucial roles during virus replication process. They regulate genes involved in viral growth and immune response processes such as Toll Like Receptor 4 signaling pathways. By comparing these proteins with their corresponding mature ones they may be useful diagnosing various conditions like chronic lymphocyte leukemia (CLL), primary effusion choriography, etc., making it possible to develop novel therapies against CML and related disorders.

Problems solved by technology

This patent discusses various methods for detecting and treating liver conditions such as viral hepatic failure syndrome(LHS). Current methodologies involve invasive procedures like histological examining, ultrasonography, CT scans, magnetic resonance spectroscopy, etc., making them expensive and slow compared to realistic options available during clinics. There also exist non-biodegradable materials called albumins, which may cause harm if taken too frequently due to their low bioavailability.

Method used

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  • Serum/plasma miRNA composition and use thereof
  • Serum/plasma miRNA composition and use thereof
  • Serum/plasma miRNA composition and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preliminary screening of specific miRNA expression profile in patients with chronic hepatitis B (Solexa sequencing entrusted Shenzhen Huada Gene Research Institute to complete)

[0067] Using Solexa sequencing technology to discover and prove that there are 88 differentially expressed microRNAs in the serum / plasma of 30 healthy controls and 30 chronic hepatitis B patients. The specific steps are:

[0068] (1) Collect serum / plasma from healthy controls and chronic hepatitis B patients;

[0069] (2) Take 80-100ml of serum respectively and add an equal volume of Trizol Reagent;

[0070] (3) Phase separation: place at room temperature for 15 minutes, then add chloroform according to the volume ratio of 0.2ml chloroform / 1ml Trizol Reagent, shake for 15s in the education column, 15 minutes at room temperature, 12,000g, 4°C, centrifuge for 15 minutes;

[0071] (4) Transfer the aqueous phase to a new 50ml centrifuge tube, and remove the protein phase in 3 steps of p...

Embodiment 2

[0085] Example 2 Real-time PCR verification was performed on the miRNAs initially screened by Solexa technology, and a group of miRNAs with stable and significant differences in expression were screened out

[0086] qRT of microRNAs was performed on sera from 30 healthy controls (Solexa sequencing samples), 30 virus-cleared individuals, 30 asymptomatic HBV-infected patients, 30 chronic hepatitis B patients (Solexa sequencing samples), and 30 chronic hepatitis C patients - PCR test.

[0087] (1) Preparation of cDNA samples: a) Take 500 μL of serum; b) Add an equal volume of water-saturated phenol, shake and mix, centrifuge at 13200 rpm for 3 minutes at 4°C, and take the supernatant; c) Supernatant + 1 / 2 volume (250 μL ) Phenol + 1 / 2 volume (250μL) chloroform, shake and mix, 4°C, centrifuge at 13200rpm for 3 minutes, take the supernatant; Take the supernatant as an RNA sample; e) Then obtain cDNA through RNA reverse transcription reaction. The reverse transcription reaction sy...

Embodiment 3

[0089] After the samples in Example 3 were reintegrated and the sample size was enlarged, the group of miRNAs screened in Example 2 was verified by quantitative PCR method, and the application and clinical value of this group of miRNAs in the process of HBV positive diagnosis were evaluated.

[0090] Serum of 100 cases of control group (50 cases of healthy controls and 50 cases of virus clearance), 75 cases of HBV group (25 cases of HBV carriers and 50 cases of chronic hepatitis B patients) and 18 cases of patients with chronic hepatitis C group were tested for microRNA. qRT-PCR test. The experimental method, qRT-PCR result processing method and clustering method are the same as in Example 2.

[0091]The results of qRT-PCR further proved that there is no significant difference between healthy controls and virus cleared persons, between HBV carriers and chronic hepatitis B patients. hsa-miR-122, hsa-miR-423-5p, hsa-miR-92a, hsa-let-7c, hsa-miR-23a, hsa-miR-23b, hsa-miR-223, hs...

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Abstract

The invention belongs to the field of molecular biology and discloses a serum/plasma miRNA composition and use thereof. The miRNA composition comprises one or more of the following miRNAs: hsa-miR-122, hsa-miR-92a, hsa-let-7c, hsa-miR-23a, hsa-miR-23b, hsa-miR-223, hsa-miR-342-3p, hsa-miR-423-5p, hsa-miR-375, hsa-miR-99a, hsa-miR-150, hsa-miR-125b and hsa-miR-10a. The miRNA composition screened out by the invention and the primers of the miRNA composition have much higher specificity and flexibility for detecting HBV-positive people and HBV-positive patients with hepatocellular carcinoma than protein markers currently used in clinic, and therefore can greatly improve the accuracy of diagnosis. The miRNA composition and the primer composition thereof both can be used in the preparation of reagents and tools for detecting HBV infection and/or HBV-positive hepatocellular carcinoma.

Description

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Claims

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Application Information

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Owner NANJING UNIV
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