Method for screening and identifying genes controlled by miRNAs

A gene and genome technology, applied in the field of screening and identification of genes regulated by miRNAs, can solve the problems of heavy workload, high false positive rate, and difficulty in finding miRNAs.

Inactive Publication Date: 2014-10-08
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the complementary binding between miRNAs and target genes can be through incomplete base matching, one miRNA may regulate many kinds of target genes, so it is very difficult to find miRNAs
At present, the search for miRNAs target genes is mainly through the prediction of biological software, but the prediction of biological software has defects such as unreliability, high false positive rate, and too much workload. Therefore, experimental methods are used to directly isolate and screen the targets regulated by miRNAs. Genes have a very important value

Method used

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  • Method for screening and identifying genes controlled by miRNAs
  • Method for screening and identifying genes controlled by miRNAs
  • Method for screening and identifying genes controlled by miRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 miRNA biomarker and transfection

[0017] The 3' end of the antisense strand of miRNA-373 was biotin-labeled, transfected into cells using the transfection reagent Lipofectamine 2000, and the total RNA was extracted using Trizol 24 hours later. After reverse transcription into cDNA, the real-time quantitative PCR method was used to detect the regulatory effect of miRNA-373 on the expression of the target gene E-cadherin, and the amplification primers used were:

[0018] E-cadherin RTF: 5'-CCTGGGACTCCACCTACAGA-3',

[0019] E-cadherin RTR: 5'-GGATGACACAGCGTGAGAGA-3'.

[0020] actinRTF: 5'-CGTGGACATCCGCAAAGAC-3',

[0021] actinRTR: 5'-TCGTCATACTCCTGCTTGCTG-3'.

[0022] Biotinylated miRNA-373 sequence:

[0023] miRNA-373anti-3biotin: 5'-GAAGUGCUUCGAUUUUGGGGUGU-3'-biotin.

Embodiment 2

[0024] Example 2 Cell culture and cell transfection

[0025] Normal breast cancer cell MCF-7 was from ATCC Company, USA. Culture in DMEM complete medium (plus 10% newborn bovine serum, 2 mM L-glutamic acid, penicillin and streptomycin) in a 37°C, 5% CO2, 90% relative humidity incubator until the cell density reaches 50 %-60%. Biotin-labeled miRNA-373 was transfected into MCF-7 cells with Lipofectamine 2000 transfection reagent. Before transfection, the medium was replaced with serum-free and antibiotic-free medium, and after 6 hours of transfection, it was changed into medium with serum and antibiotics to continue culturing.

Embodiment 3

[0026] Example 3 Streptavidin-Biotin pull-down experiment

[0027] Biotin-labeled miRNA-373 was transfected into MCF-7 cells with liposome Lipofectamine2000 as above, and 270 μL of 37% formalin solution (final concentration of 1 %) at room temperature for 10 min to fully cross-link proteins and RNA. Then add 1mL 10x Glycine (glycine) at room temperature for 5min to terminate the reaction. Aspirate the liquid in the culture dish, wash the cells twice with 10mL pre-cooled 1xPBS, scrape the cells with 1mL 1xPBS (2% Cocktail, RNase Inhibitor 100U / mL), harvest the cells at 800g, 3min in a 1.5mL RNase-Free centrifuge tube middle. Add 500 μL lysis buffer to each tube of cells, mix well, and let stand on ice for 30 minutes. Ultrasonic treatment was performed to break up the genomic DNA. Ultrasound working conditions: 200W, 5 seconds of ultrasound, 5 seconds of interval, 15 times of ultrasound. After standing on ice for 5 minutes, centrifuge at 14000g for 30s at 4°C. Transfer the ...

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Abstract

The invention provides a method for directly screening and identifying genomic DNA (Deoxyribose Nucleic Acid) controlled by miRNAs (micro Ribose Nucleic Acids). The method comprises the steps of marking the studied target miRNAs with a biotin, separating and purifying the genomic DNA which is in targeted combination with the biotin marked miRNAs in the physiological status by use of the affinity between the biotin and a streptavidin, next, performing cloning and sequencing on the separated and purified DNA to obtain DNA sequence information, and analyzing the positions and names of genes of the DNA sequence by use of the bioinformatics, thereby obtaining all the target genes of the miRNAs controlling the genomic DNA. The method can be applied to the functional study of the miRNAs of all targeted genomic DNAs.

Description

[0001] technical field [0002] The invention relates to a method for screening and identifying genes regulated by miRNAs, and provides a simple and practical method for finding possible target genes regulated by miRNAs in functional research. Background technique [0003] miRNAs are a class of single-stranded RNA molecules composed of about 19-23 bases, which are small RNAs that cannot encode proteins but have important regulatory functions. Mature miRNAs can bind to related sequences of target genes through complementary base pairing. It is now known that the reported binding sites can be located in the 3' and 5' untranslated regions of gene mRNA, and may also be located in the gene promoter region. It is now known that miRNAs have important regulatory functions in various life activities including cell differentiation, apoptosis, proliferation, development, hematopoiesis and tumors. Because miRNAs play a role in regulating life activities by regulating the expression of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6818
Inventor 向双林魏科颜峰胡翔张健
Owner HUNAN NORMAL UNIVERSITY
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