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Micro ribonucleic acid (RNA), and use of precursor and antisense nucleic acid thereof in regulation of catalase activity

A technology of catalase and RNA precursor, which is applied in the professional field of biomedicine and can solve problems such as increased expression

Active Publication Date: 2014-06-11
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

sod-5 and sod-3 encode copper-zinc SOD and manganese SOD, respectively, but the isoforms encoded by these two genes are relatively small and only play an auxiliary supplementary role, and the expression is significantly increased at the dauer larval stage

Method used

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  • Micro ribonucleic acid (RNA), and use of precursor and antisense nucleic acid thereof in regulation of catalase activity
  • Micro ribonucleic acid (RNA), and use of precursor and antisense nucleic acid thereof in regulation of catalase activity
  • Micro ribonucleic acid (RNA), and use of precursor and antisense nucleic acid thereof in regulation of catalase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning and analysis of embodiment 1miRNA

[0041] 1. Extraction and purification of RNA

[0042] Get 20 wild-type Caenorhabditis elegans, 20 SEQ1 deletion nematodes SEQ1 (- / -) respectively placed in 40ulM9 buffer (KH 2 PO 4 3g; Na 2 HPO 4 6g; NaCl5g; 1MMgSO 4 1ml; add water to make up to 1000ml. ) into a RNase-free 1.5ml centrifuge tube (purchased from Axygen), add 500ul of total RNA extraction reagent Trizol (purchased from Invitrogen), vortex for 30 seconds to mix, and ice-bath for 30 minutes to dissolve the parasites. This was followed by centrifugation at 12000 rpm for 10 minutes at 4°C. Transfer the supernatant to another RNase-free 1.5ml centrifuge tube, add 100ul chloroform, vortex for 30 seconds, and place at room temperature for 3 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C. Transfer the supernatant to another RNase-free centrifuge tube. Add an equal volume (usually about 300ul) of isopropanol and mix well. Let stand at room temperature fo...

Embodiment 2

[0058] Embodiment 2 Real-time quantitative fluorescent PCR detects the expression of SEQ1 microRNA in nematode strains

[0059] Express

[0060] Wild-type Caenorhabditis elegans and SEQ1-deficient nematode strain SEQ1(- / -) were cultivated, and nematode RNA was extracted and reverse-transcribed according to the method in Example 1. After reverse transcription, the expression difference of SEQ1 between wild type and SEQ1(- / -) was detected by real-time quantitative fluorescent PCR (RT-PCR).

[0061] The SYBRPremixExTaq kit (purchased from TAKARA) was used according to the manufacturer's product instructions. Add 2ul template, 10ul2×SYBRPremixExTaq, 0.4ul forward primer (5'-GTGCAGGGTCCGAGGT-3') (SEQ.ID.No.4) 10uM, 0.4ul reverse primer (5'- AGGCAAGATGTTGGCA-3') 10uM, 0.4ul 50×ROXReference dye (included in the SYBRPremixExTaq kit), add sterilized distilled water to 20ul.

[0062] Use U6 as an internal reference, refer to the calculation method of QuantitativeReal-TimeRT-PCR of Th...

Embodiment 3

[0064] Example 3 Real-time quantitative fluorescent PCR detects the expression of catalase in each strain of nematode

[0065] Wild-type C. elegans and the SEQ1-deficient nematode strain SEQ1(- / -) were cultivated, and nematode RNA was extracted and reverse-transcribed according to the method in Example 1.

[0066] ctl-1 reverse transcription primer: 5'-GAGCTGATGGCGAAGAGC-3' (SEQ.ID.No.9)

[0067] ctl-2 reverse transcription primer: 5'-TGGATGAGTGCCTTGACA-3' (SEQ.ID.No.10)

[0068] After reverse transcription, real-time quantitative fluorescent PCR was used to detect the expression difference of ctl-1 and ctl-2 between wild type and SEQ1(- / -). The experimental conditions are the same as in Example 2.

[0069] Primers used in real-time quantitative fluorescent PCR reaction

[0070] ctl-1RT upstream primer: 5'-CGCATCTCCCTGGCTTTCAT-3' (SEQ.ID.No.11)

[0071] ctl-1RT downstream primer: 5'-CTCGCCAGACAAGATGCTCC-3' (SEQ.ID.No.12)

[0072] ctl-2RT upstream primer: 5'-ATCATGACATTCCC...

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Abstract

The invention provides a micro ribonucleic acid (RNA), and the use of a precursor and an antisense nucleic acid thereof in the regulation of catalase activity. The micro RNA provided by the invention comprises nucleotide or a bioactivity functional segment thereof or a variant thereof with the following sequence: 5'-AGGCAAGAUGUUGGCAUAGCUGA-3'. Experiments show that through expressing the micro RNA or the precursor thereof, the catalase activity can be reduced, and through using the antisense nucleic acid of the micro RNA to inhibit the expression of the micro RNA or the precursor thereof, the catalase activity can be increased.

Description

technical field [0001] The invention belongs to the professional field of biomedicine, and relates to microRNA (miRNA), its precursor and its antisense oligonucleotide sequence for regulating the activity of catalase, and their role in the prevention and treatment of cardiovascular diseases and a series of elderly patients. Uses in diseases and in reducing inflammatory responses. Background technique [0002] MicroRNA (microRNA, miRNA) is a single-stranded non-coding RNA with a length of about 22nt, which regulates gene expression by affecting the stability of messenger RNA. miRNA was first discovered in C. elegans. In 1993, Lee et al. found that lin-4 regulated the translation of lin-14 through complementary RNA-RNA interaction, and ultimately affected the development of C. elegans. [0003] So far, miRNAs have been found in various species, and their functions are constantly being revealed. These miRNAs widely exist in various organisms and participate in cell proliferat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85
Inventor 李培峰周露玙丁素玲王昆
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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