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Application of circRNA in the preparation of diagnostic reagents 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: 2021-06-29
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 the preparation of diagnostic reagents for proliferative vitreoretinopathy
  • Application of circRNA in the preparation of diagnostic reagents for proliferative vitreoretinopathy
  • Application of circRNA in the preparation of diagnostic reagents 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] Step 1: Sample preparation: Epiretinal membrane specimens (experimental group, n=30) and cataract proliferative membrane specimens (control group, n=30) after ophthalmic vitreous surgery, total RNA was extracted with TRIzol (Invitrogen) reagent, and stored Store at -80°C for later use.

[0055] Step 2: Differential expression screening:

[0056] The expression profiling chip of Agilent Company in the United States was used to analyze the circRNA related to the occurrence of PVR disease; the specific steps of analysis were as follows: the experimental sample RNA was used Agilent expression profiling chip supporting kit, Low Input Quick Amp WTLabeling Kit (Cat.# 5190-2943) and standard The operating procedure is to amplify and label the total RNA of the sample, and use the RNeasy mini kit (Cat.# 74106, QIAGEN) to purify the labeled cRNA; follow the hybridization st...

Embodiment 2

[0060] Embodiment 2: preparation kit of the present invention

[0061] 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 Sangon Co., Ltd., with a purity of PAGE grade. The synthesized primers were dissolved in DEPC H2O with a total concentration of 10 μM. The internal reference primer was 18 S rRNA.

[0062] Prepare a kit comprising the following components:

[0063] (a) Extraction system:

[0064] 1) Trizol reagent, 1 tube, 5000 μL / tube;

[0065] 2) Chloroform, 1 tube, 1000 μL / tube;

[0066] 3) Absolute ethanol, 1 tube, 10000 μL / tube;

[0067] 4) DEPC ddH 2 O, 1 tube, 10000 μL / tube;

[0068] 5)ddH 2 O, 1 tube, 10000 μL / tube;

[0069] 6) Isopropanol, 10000 μL / tube;

[0070] (b) Reverse transcription system:

[0071] 1) Total RNA reverse transcription primer (Random 6 mers), 1 tube, concentration: 50 μM, 100 μL / tube;

[0072] 2) Reverse transcriptase (200 U / μL) 100 μL; ...

Embodiment 3

[0083] Embodiment 3: kit detection of the present invention

[0084] 1. Separation of serum

[0085] 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.

[0086] 2. RNA Extraction of Serum and Vitreous Samples

[0087]Using the total RNA extraction system described in 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 stored at -80°C for a long time). Add 200 μl of chloroform to every 1 ml of TRIzol, vibrate vigorously and mix well, then place at room temperature for 3-5 min to allow natural phase separation. Centrifuge at 12,000 rpm for 15 min at 4°C. The sample will separate into three layers: a yellow organic pha...

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Abstract

The invention discloses the application of circRNA in the preparation of proliferative vitreoretinopathy (PVR) diagnostic reagents. The invention also discloses a circRNA detection kit for PVR diagnosis. The kit mainly includes RNA extraction system, reverse transcription reaction system and PCR reaction system. The relative content of circRNA in vitreous and serum was detected by real-time quantitative PCR technology for early diagnosis of PVR. This method has the characteristics of less trauma, 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 proliferative vitreoretinopathy (PVR) diagnostic reagents, a circRNA kit for assisting the early diagnosis of PVR disease, 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 a major cause of failed retinal reattachment surgery and retinal re-detachment, leading to severe impairment of visual function and even blindness. PVR is common in diseases such ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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