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Novel circRNA and application thereof

A sequence and reagent technology, applied in the field of disease detection, can solve the problems of lack of effective methods for early diagnosis and achieve good application prospects, good stability, fast and accurate quantitative results

Active Publication Date: 2019-06-21
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective method for the early diagnosis of the disease

Method used

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  • Novel circRNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The establishment and sampling of the hypoxia-ischemia rat model of embodiment 1

[0035] 7-day-old neonatal SD rats with a body weight of 10-14 g. They were randomly divided into two groups, a sham operation group (normal control group) and a hypoxic-ischemic group, with 3 rats in each group. Under ether anesthesia, a median neck incision was made, and the left common carotid artery was separated. The sham operation group did not receive any treatment; the operation group ligated and cut off the left common carotid artery, rested for 1 hour, and placed in 8% oxygen and nitrogen mixed gas for 2 hours. One day after the experiment, the rats in each group and at each time point were decapitated under ether anesthesia, and the brain tissues were taken out and immediately put into liquid nitrogen for cryopreservation.

Embodiment 2

[0036] Example 2 High-throughput sequencing and data analysis

[0037] 2.1 Extraction and quality inspection of tissue total RNA

[0038] (1) Cut the tissues of each group into pieces on ice, take an appropriate amount (50-100 mg) of the tissues, add 1 ml of pre-cooled Trizol to the EP tube without RNase, pipette evenly, and let stand on ice for 10 minutes;

[0039] (2) Add 200 μl of pre-cooled chloroform, shake vigorously for 30 sec, then let stand on ice for 15 min, and centrifuge at 14000 rpm / min at 4°C for 20 min;

[0040] (3) Take out the supernatant and put it in a new RNase-free EP tube (be careful not to touch the middle layer), add an equal volume of pre-cooled isopropanol (about 500 μl), mix well and let stand on ice for 0.5-1h, 4 Centrifuge at 14000rpm / min for 20min;

[0041] (4) Discard the supernatant, add 1 ml of pre-cooled 75% ethanol, mix upside down, and centrifuge at 14000 rpm / min at 4°C for 20 min;

[0042] (5) Discard the supernatant, add 1 ml of pre-coo...

Embodiment 3

[0062] Example 3 RT-PCR verification of the relative expression of circRNA in the control group and the model group

[0063] 1. Model building

[0064] Concrete method is with embodiment 2. The sham operation group (normal control group) and the hypoxia-ischemia group had 20 rats in each group.

[0065] 2. Experimental method

[0066] 2.1 Extraction of total tissue RNA

[0067] Reference Example 2

[0068] 2.2 Primer design

[0069] For the circular closed structure of circRNA, design back-to-back primers (divergent primer), GAPDH is used as an internal reference, and sent to a primer synthesis company for synthesis.

[0070] circRNA primers:

[0071] Upstream primer: 5'-CAGTCAGATGGTGGTTCATTTCA-3' (SEQ ID NO.2)

[0072] Downstream primer: 5'-GGTCATAGAAACCCAAAAGTGC-3'(SEQ ID NO.3)

[0073] Target gene amplification length: 145bp.

[0074] 2.3 Reverse transcription

[0075] According to PimeScript TM RT reagent Kit (Perfect Real Time) RR037A (Takara) manual configure...

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Abstract

The invention relates to novel circRNA and application thereof, particularly application thereof in preparing hypoxic-ischemic brain damage diagnosis reagents. Existing studies display that the circRNA plays an important role in occurrence and development of diseases and is expected to become a biomarker of diseases like cancer, heart disease, nervous system disease and arteriosclerosis. The novelcircRNA has high relevance with hypoxic-ischemic brain damage and has quite important study value and clinical application value.

Description

technical field [0001] The present invention belongs to the field of disease detection, specifically relates to a new circRNA and its application, and more specifically relates to the application of the new circRNA in the preparation of a diagnostic reagent for hypoxic-ischemic brain injury. Background technique [0002] Circular RNA (circular RNA, circRNA), a new type of endogenous non-coding RNA, is a recent research hotspot in the field of RNA. CircRNA is a type of circular RNA molecule formed by connecting the 3' and 5' ends by back splicing . According to different splicing sources, circRNAs can be divided into intron-derived circRNAs and exon-derived circRNAs. circRNA has the following characteristics: 1) It belongs to non-coding RNA; 2) It is enriched in the cytoplasm, forming a loop and easy to exit the membrane; 3) It has a specific reverse splicing site; 4) It is stable and not easily degraded by exonucleases; 5 ) has certain tissue specificity, timing specificit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6883
Inventor 杨丽君崔红赵贺华张囡
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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