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Application of circRNA in preparation of diagnostic reagent for proliferative vitreoretinopathy

A vitreoretinal and diagnostic reagent technology, applied in the field of medical biological detection, can solve the problem of no early diagnostic markers for circRNA proliferative vitreoretinopathy, and achieve the effect of less trauma and simple operation

Active Publication Date: 2019-02-19
EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there is no research on circRNA as an early diagnostic marker for proliferative vitreoretinopathy

Method used

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  • Application of circRNA in preparation of diagnostic reagent for proliferative vitreoretinopathy
  • Application of circRNA in preparation of diagnostic reagent for proliferative vitreoretinopathy
  • Application of circRNA in preparation of diagnostic reagent for proliferative vitreoretinopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Verification of the correlation between circ_0043144 and proliferative vitreoretinopathy

[0054] 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.

[0055] Step 2: Differential expression screening:

[0056] The circRNA related to the occurrence of PVR disease was analyzed by using the expression profiling chip of Agilent company in the United States; the specific steps of analysis were as follows: the experimental sample RNA was matched with Agilent expression profiling chip kit, Low Input Quick Amp WT LabelingKit (Cat.#5190-2943) and standard The operating procedure is to amplify and label the total RNA of the sample, and purify the labeled cRNA with RNeasymini kit (Cat. .#5188-5242), in the rolling hybridizatio...

Embodiment 2

[0061] Embodiment 2: preparation kit of the present invention

[0062] The sequence of circ_0043144 is shown in SEQ ID: NO:1. The upstream and downstream primers for specific quantitative PCR were synthesized by Shanghai Shenggong Company, with a purity of PAGE grade. The synthesized primers were synthesized using DEPC H 2 O was dissolved, the total concentration was 10 μM, and the internal reference primer was 18S rRNA.

[0063] Prepare a kit comprising the following components:

[0064] (a) Extraction system:

[0065] 1) Trizol reagent, 1 tube, 5000 μL / tube;

[0066] 2) Chloroform, 1 tube, 1000 μL / tube;

[0067] 3) Absolute ethanol, 1 tube, 10000μL / tube;

[0068] 4) DEPC ddH 2 O, 1 tube, 10000 μL / tube;

[0069] 5)ddH 2 O, 1 tube, 10000 μL / tube;

[0070] 6) Isopropanol, 10000 μL / tube;

[0071] (b) Reverse transcription system:

[0072] 1) Total RNA reverse transcription primer (Random 6mers), 1 tube, concentration: 50 μM, 100 μL / tube;

[0073] 2) Reverse transcrip...

Embodiment 3

[0084] Embodiment 3: kit detection of the present invention

[0085] 1. Separation of serum

[0086] The blood samples of the examined individual and the healthy individual used as a reference are collected, and the serum and blood cells are separated from the heparin anticoagulated tube by centrifugation for the detection of the disease. The centrifugation conditions were 4°C, 12,000 rpm, 10 min.

[0087]2. The RNA extraction of serum and vitreous samples adopts the total RNA extraction system of the present invention. After adding TRIzol, 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 at -80°C for a long time) save). 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 min. The sample will separate into three layers: a yellow organic phase, ...

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Abstract

The invention discloses an application of circRNA in preparing a diagnostic reagent for proliferative vitreoretinopathy (PVR). The invention also discloses a circular RNA detection kit for PVR diagnosis. The kit mainly comprises an RNA extraction system, a reverse transcription reaction system, and a PCR reaction system. The relative contents of circRNA in vitreous and serum are detected by real-time fluorescence quantitative PCR technique for early diagnosis of PVR. The application of circRNA in preparing the diagnostic reagent for PVR has the advantages of small wound, high sensitivity and simple operation.

Description

technical field [0001] The invention belongs to the technical field of medical biological detection, and specifically relates to the application of circRNA in the preparation of diagnostic reagents for proliferative vitreoretinopathy (PVR), a circRNA kit for assisting the early diagnosis of PVR diseases, and its use in assisting the early diagnosis of PVR Applications and detection methods. Background technique [0002] Proliferative vitreoretinopathy (PVR) is a complex pathological reaction composed of a variety of cellular components, vitreous, extracellular matrix, and a large number of autocrine or paracrine cytokines. A disorder in which the extensive fibrous proliferative membranes on the surface of the retina and behind the vitreous shrink and stretch, causing retinal detachment. PVR has become the main cause of retinal reattachment failure and retinal detachment, which can lead to severe impairment of visual function and even blindness. PVR is common in diseases su...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6851A61K31/7088A61P9/10A61P27/02
CPCA61K31/7088A61P9/10A61P27/02C12Q1/6851C12Q1/6883C12Q2600/158C12Q2600/178C12Q2531/113C12Q2563/107
Inventor 颜标蒋沁赵晨李秀苗单琨周荣妹
Owner EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV
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