Application of lncrna-malat1 in the preparation of diagnostic reagents for proliferative vitreoretinopathy

A vitreoretinal and proliferative technology, applied in the field of medical biological detection, can solve the problems of proliferative vitreoretinopathy without lncRNA-MALAT1, and achieve the effect of less trauma and strong operability

Active Publication Date: 2018-08-28
南京医科大学眼科医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Currently, there is no report of lncRNA-MALAT1 as a biomarker for the early diagnosis of proliferative vitreoretinopathy

Method used

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  • Application of lncrna-malat1 in the preparation of diagnostic reagents for proliferative vitreoretinopathy
  • Application of lncrna-malat1 in the preparation of diagnostic reagents for proliferative vitreoretinopathy
  • Application of lncrna-malat1 in the preparation of diagnostic reagents for proliferative vitreoretinopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Verification of the correlation between lncRNA-MALAT1 and proliferative vitreoretinopathy

[0063] (1) lncRNA microarray analysis and screening of lncRNA related to PVR disease and verification, the specific process is as follows figure 1 shown.

[0064] The first step: sample preparation: epiretinal membrane specimens (experimental group, n=30) and cataract proliferation membrane specimens (control group, n=30) after ophthalmic vitreous surgery, total RNA was extracted with TRIzol (Invitrogen) reagent, and preserved Store at -80°C for later use.

[0065] Step 2: Differentially expressed lncRNA screening:

[0066] Using the lncRNA expression profile chip of Aglient Company in the United States, the lncRNA related to the occurrence of PVR disease was analyzed; the specific steps of the analysis were as follows: the fluorescent group was used to label the lncRNA with a labeling enzyme to obtain a fluorescent probe for hybridization with the chip, and MAUI was ...

Embodiment 2

[0071] Embodiment 2: preparation kit of the present invention

[0072] The sequence of MALAT1 is shown in SEQ ID: NO: 1, its specific quantitative PCR upstream and downstream primers and internal reference GAPDH and / or Beta-tubulin quantitative PCR upstream and downstream primers, designed by Primer5, is responsible for primer synthesis by Invitrogen Company, the purity is PAGE grade, synthesized primers using DEPC H 2 O was dissolved to a total concentration of 10 μM.

[0073] Prepare a kit comprising the following components:

[0074] (a) Extraction system

[0075] 1) Trizol reagent, 1 tube, 2000 μL / tube;

[0076] 2) Chloroform, 1 tube, 500 μL / tube;

[0077] 3) Absolute ethanol, 1 tube, 8000 μL / tube;

[0078] 4) DEPC ddH2O, 1 tube, 1000 μL / tube;

[0079] 5) ddH2O, 1 tube, 2000 μL / tube;

[0080] 6) Isopropanol, 8000 μL / tube;

[0081] (b) Reverse transcription system

[0082]1) Total RNA reverse transcription primers (including Oligo dT and Random6mers), 1 tube, conce...

Embodiment 3

[0096] Embodiment 3: kit detection of the present invention

[0097] 1. Separation of plasma and blood cell samples

[0098] The blood samples of the examined individuals and their reference healthy individuals were collected, and the serum and blood cells were separated by centrifugation using heparin anticoagulant tubes for the detection of lncRNA. The centrifugation conditions were 4°C, 12,000 rpm, 10 min.

[0099] 2. RNA Extraction from Plasma and Blood Cell Samples

[0100] In the separated blood cells and plasma samples, use the total RNA extraction system of the kit of the present invention, add TRIzol and place it at room temperature for 10 minutes to fully lyse the sample (note: if the next step is not performed, the sample can be placed in- long-term storage at 70°C). Add 200 μl of chloroform to every 1ml of TRIzol, vibrate vigorously and mix well, then place at room temperature for 3-5min to allow natural phase separation. Centrifuge at 12,000 rpm at 4°C for 15 ...

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Abstract

The invention provides application of lncRNA-MALAT1 in preparing a proliferative vitroretinopathy (PVR) diagnosis reagent. The invention also provides an MALAT1 detection kit for PVR diagnosis. The kit comprises an RNA (ribonucleic acid) extraction system, a reverse transcription reaction system and a PCR (polymerase chain reaction) reaction system. On the basis of real-time fluorescent quantitative PCR, the kit can be used for carrying out PVR early diagnosis by detecting the relative content of MALAT1 in the serum or plasma. The method has the characteristics of small trauma and the like, and is convenient to operate.

Description

technical field [0001] The invention belongs to the technical field of medical biological detection, and specifically relates to the application of lncRNA-MALAT1 in the preparation of diagnostic reagents for proliferative vitreoretinopathy, a MALAT1 kit for assisting the early diagnosis of PVR diseases, and its application in assisting the early diagnosis of PVR and detection methods. Background technique [0002] Proliferative vitreoretinopathy (PVR) is commonly seen in excessive condensation, electrocoagulation, trauma, giant retinal breaks, multiple retinal breaks, long-term rhegmatogenous retinal detachment, multiple intraocular operations, ocular trauma, and intraocular inflammation. Its potential risk factors are not very sure, and the pathogenesis is very complicated. Therefore, the early diagnosis of PVR disease is still one of the most severe challenges facing ophthalmologists. However, so far, there is no specific biological index for the auxiliary diagnosis of PV...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6851A61K31/7088A61P27/02
CPCA61K31/7088C12Q1/6883C12Q2600/158C12Q2600/178
Inventor 颜标蒋沁姚进周荣妹
Owner 南京医科大学眼科医院
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