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circRNA and application thereof in detecting hypoxic-ischemic brain damage

A technology for hypoxia-ischemia and brain injury, which is applied in the field of disease detection, can solve the problems of unclear diagnosis of brain injury and no effective treatment methods, and achieve the effect of accurate detection method and high detection accuracy

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

④Translational protein: Although circRNA has always been considered as non-coding RNA, recent studies have shown that some circRNA may have a translation function
Unfortunately, the diagnosis of hypoxic-ischemic brain injury is still unclear, and there is no effective treatment

Method used

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  • circRNA and application thereof in detecting hypoxic-ischemic brain damage
  • circRNA and application thereof in detecting hypoxic-ischemic brain damage
  • circRNA and application thereof in detecting hypoxic-ischemic brain damage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The establishment and sampling of the hypoxia-ischemia rat model of embodiment 1

[0036] 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

[0037] Example 2 High-throughput sequencing and data analysis

[0038] 2.1 Extraction and quality inspection of tissue total RNA

[0039] (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;

[0040] (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;

[0041] (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;

[0042] (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;

[0043] (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'-CCACAGCCACAAACCCTTTT-3' (SEQ ID NO.2)

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

[0073] Target gene amplification length: 95bp.

[0074] 2.3 Reverse transcription

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

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Abstract

The invention relates to circRNA and an application thereof in detecting hypoxic-ischemic brain damage. Aiming at the problem that there is no effective diagnosis method for hypoxic-ischemic brain damage at present, the inventor adopts a high-throughput sequencing technology to sequence the circRNA transcriptome of the hypoxic-ischemic brain damage model and uses a bioinformatics method to analyze, so as to find circRNA related to the hypoxic-ischemic brain damage, the circRNA provides a new molecular diagnosis marker for the diagnosis of the hypoxic-ischemic brain damage, and provides a research basis for clinical detection.

Description

technical field [0001] The invention belongs to the field of disease detection, and in particular relates to circRNA and its application in the preparation of diagnostic reagents for hypoxic-ischemic brain injury. Background technique [0002] In recent years, circular RNA (circular RNA, circRNA) has become another non-coding RNA research hotspot after miRNA and 1ncRNA, and has attracted extensive attention from scholars at home and abroad. At present, the research on the biological function of circRNA is still in the exploratory stage, and its mechanism of action mainly includes the following four types: ① miRNA sponge effect: ecircRNA located in the cytoplasm can bind miRNA to prevent it from binding to downstream target genes through a competitive endogenous RNA mechanism , thereby regulating gene expression. The miRNA sponge effect is currently the most studied mechanism of circRNA action in disease. ②Regulation of transcription and splicing: Some circRNAs can affect t...

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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