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PiRNA combination for detecting heart disease, and application thereof

A technology for heart disease and heart tissue, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial determination/inspection, etc.

Active Publication Date: 2017-07-04
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the level of small RNA expression regulation, although the research on microRNA has achieved certain results, there are still many unsolved problems, and its mechanism needs to be further elucidated, while piRNA has many potential functions that need to be explored

Method used

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  • PiRNA combination for detecting heart disease, and application thereof
  • PiRNA combination for detecting heart disease, and application thereof
  • PiRNA combination for detecting heart disease, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Rapid screening of differentially expressed hearts under the ischemia-reperfusion model by high-throughput sequencing piRNA

[0089] Take C57 mature male mice more than 8 weeks old, and make myocardial infarction model according to the method of our laboratory, that is, the left anterior descending branch of the left ventricle of the mouse is ligated after thoracotomy to make the left ventricle ischemia for 45 minutes, then loosen the ligation, and reperfuse After 3 hours, observe and determine the infarction of the myocardium. The infarcted tissue and normal control heart tissue were quickly placed in liquid nitrogen and sent to Shenzhen Huada Gene Company for further RNA extraction, detection and sequencing. The sequencing method used is high-throughput Solexa deep sequencing, which can obtain a large amount of small RNA information in a short time. The small RNA (sRNA) obtained by Solexa deep sequencing covers almost all RNAs, including miRNA, siRNA, p...

Embodiment 2

[0099] Example 2 Using fluorescent quantitative PCR method to detect the expression of piRNA in primary cardiomyocytes

[0100] The specific steps for the preparation of primary cardiomyocytes are as follows: the heart of a 2-day-old C57 mouse was peeled off, chopped and put into phosphate buffer (1L distilled water contained 8g of NaCl, 0.2g of KCl, anhydrous Na 2 HPO 4 1.44g, KH 2 PO 4 0.24g), then at 37°C, HEPES-buffered saline solution (20mM / L HEPES-NaOH, pH7.6, 130mM / L NaCl, 3mM / LKCl, 1mM / LNaH 2 PO 4 , 4mM / L glucose and 0.15% trypsin by weight / volume ratio), after centrifugation, the cells were resuspended in heat-inactivated horse serum containing 5% by volume, 100mM / L ascorbic acid, 1mg / mL transferrin, 10ng / mL Selenium, 100U / mL penicillin, 100U / mL streptomycin in DMEM / F12 cell culture medium. The isolated cardiomyocytes were cultured at 37°C for 1 hour, and then diluted to 1×10 6 The cells / mL were spread in a culture dish, and 0.1mM / L 5-bromodeoxyuridine was add...

Embodiment 3

[0109] Example 3 Detection of piRNA Expression in Normal Heart and Myocardial Infarction Heart Using Fluorescent Quantitative PCR Condition

[0110] Total RNA was extracted from normal heart tissue and myocardial infarction heart tissue, and 1 μg of the extracted RNA was reverse-transcribed with the participation of RNA reverse transcriptase RTAce (Toyobo) using the corresponding reverse transcription primers (Table 5, RT-PH). Reverse transcription in a constant temperature water bath at 42°C for 1 hour, denaturation at 99°C for 5 minutes, see Example 2 for the specific reverse transcription process. The reverse transcription product was detected by fluorescent quantitative PCR using PH-F and loop-R in Table 5. See Example 2 for the specific process. Here we have optimized four piRNAs, SEQ ID NO7, SEQ ID NO12, SEQ ID NO 15, and SEQ ID NO16, namely piR-7, piR-12, piR-15, and piR-16, respectively, and the corresponding fluorescent PCR probes were respectively For SEQ ID NO1...

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PUM

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Abstract

The invention provides a piRNA combination for detecting the heart disease, and an application thereof. The piRNA combination comprises one or more of 16 RNA sequences represented by SEQ ID No. 1 to NO.16. The application of the piRNA combination is the application of the piRNA combination in the preparation of medicines or kits for predicting, diagnosing or, identifying and / or treating the heart disease. The piRNA playing a key role in the regulation of myocardial ischemial injury and myocardial infarction, and a complete method is provided for researching the regulation mechanism. The research method is helpful to elucidate the pathogenesis of myocardial infarction, provides a new idea for the prevention and diagnosis of the myocardial infarction, and is of great significance to especially develop the piRNA as a medicine for treating the heart disease.

Description

technical field [0001] The invention relates to RNA for detecting heart disease, in particular to a combination of piRNA for detecting heart disease and its application, and belongs to the field of biotechnology. Background technique [0002] Myocardial infarction is a serious ischemic heart disease and a major threat to human health. Cardiovascular disease ranks first in the proportion of deaths from major diseases among Chinese residents, and it is estimated that there are at least 230 million people with cardiovascular diseases in my country. With the growth of population aging, the incidence of cardiovascular diseases in my country will increase significantly. It is estimated that in 2030, the number of patients with cardiovascular diseases in my country will increase by 21.3 million, and the number of deaths from cardiovascular diseases will increase by 7.7 million. [0003] Ischemic myocardial injury is caused by the imbalance between coronary blood flow and heart dem...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/113
CPCC12Q1/686C12Q1/6883C12Q2600/136C12Q2600/158C12Q2600/178C12Q2563/107C12Q2545/114
Inventor 李培峰王胤王建勋
Owner QINGDAO UNIV
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