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Breast cancer related microRNA (Ribonucleic Acid) detection kit

A detection kit and breast cancer technology, applied in the field of molecular biology, can solve the problems of not being able to control the cross-hybridization of probes and non-specific sequences in cells, and the difficulty of simultaneous in-situ detection of expression, etc., to achieve simple design and improved Fluorescence signal intensity, effect of improving detection sensitivity

Active Publication Date: 2017-05-10
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many difficulties in the multiple parallel detection of miRNA in the current prior art.
Firstly, labeled probes for each target miRNA need to be prepared separately; secondly, it is difficult to simultaneously detect the expression of multiple target miRNAs in situ
Therefore, the currently reported detection of multiple miRNAs can only use different labeling methods, however, the use of different labeling methods cannot well control the possible cross-hybridization of probes with non-specific sequences in cells

Method used

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  • Breast cancer related microRNA (Ribonucleic Acid) detection kit
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  • Breast cancer related microRNA (Ribonucleic Acid) detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Breast cancer related miRNA detection kit

[0033] This embodiment provides a breast cancer-related miRNA detection kit, including a capture probe and a signal amplification probe, the signal amplification probe includes a primary signal amplification probe, a secondary signal amplification probe, and a tertiary signal amplification probe. probe. The above probes have the characteristics of strong specificity and high sensitivity.

[0034] 1. Capture probe

[0035] The capture probe is a probe that connects the target nucleic acid and the primary signal amplification probe. The base sequence of each capture probe is from the 5' end to the 3' end in turn the specific sequence P1 that binds to the target nucleic acid to be detected, the spacer sequence , the P2 sequence that can be complementary to the P3 sequence of the primary signal amplification probe, and the P2 sequences for different target genes are different from each other.

[0036] The spacer is used to spac...

Embodiment 2

[0065] Example 2 A kit for detecting breast cancer-related miRNA

[0066] The invention provides a breast cancer-related miRNA detection kit, which can detect target miRNAs including: hsa-miR-148b-3p, hsa-miR-409-3p, hsa-miR-16-5p, hsa-miR The expression levels of -25-3p, hsa-miR-222-3p, hsa-miR-324-3p, hsa-miR-206 and hsa-miR-105-5p can be used in actual detection according to specific needs The corresponding P1-P8 sequences are used to form a detection kit to realize detection.

[0067] The components of the detection kit in this embodiment include: capture probes, signal amplification probes and fluorescent groups, and the specific probe components are shown in Table 7.

[0068] In this example, hsa-miR-148b-3p, hsa-miR-409-3p, hsa-miR-16-5p, hsa-miR-25-3p, hsa-miR-222-3p, hsa-miR-324- 3p, hsa-miR-206 and hsa-miR-105-5p were randomly divided into 2 groups for detection.

[0069] Table 7 is aimed at the detection kit of specific target gene (table number is SEQ ID NO.)

...

Embodiment 3

[0072] Example 3 Using the kit in Example 2 to detect the sample

[0073] In this example, the kit of Example 2 will be used to detect breast cancer cells.

[0074] The source of breast cancer cells is: breast cancer cell line MCF-7. Those skilled in the art can obtain related cell lines in existing products according to the name of the cell lines.

[0075] The formula of described various solutions is as follows:

[0076]

[0077] All the probes in the corresponding list in Example 2 were used in the signal amplification probe mixture in this example.

[0078] 1. Sample pretreatment, filter CTCs onto the filter membrane

[0079] 1. Preserve the blood sample in the sample preservation tube with preservation solution, centrifuge at 600×g for 5 minutes, and discard the supernatant.

[0080] 2. Add 4mL PBS and 1mL fixative, vortex to mix, and let stand at room temperature for 8min.

[0081] 3. Sample filtration: Transfer the liquid in the sample storage tube to the filter,...

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Abstract

The invention relates to a breast cancer related microRNA (Ribonucleic Acid) detection kit. The breast cancer related microRNA detection kit comprises at least one first-stage signal amplification probe, at least one second-stage signal amplification probe, at least one third-stage signal amplification probe and a capture probe which are provided for each kind of to-be-detected breast cancer related microRNA, wherein the P1 sequence of the capture probe of the breast cancer related microRNA is selected from SEQ ID NO. 1 to SEQ ID NO. 8. According to the breast cancer related microRNA detection kit disclosed by the invention, hybridization reaction can be carried out by the capture probe and the signal amplification probes which are selected by the breast cancer related microRNA detection kit under a uniform reaction condition, and all probes are not in nonspecific binding; the designed probes have good specificity and high signal-to-noise ratio during detection; meanwhile, a system with a good detection effect is formed by an identification kit and a detection method through combined use of multiple probes.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a breast cancer-related microRNA detection kit. Background technique [0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with regulatory functions found in eukaryotes, with a size of about 20-25 nucleotides. Mature miRNAs are produced by a series of nuclease cleavage processes from longer primary transcripts, and then assembled into the RNA-induced silencing complex (RNA-induced silencing complex, RISC), which is recognized by complementary base pairing target miRNA, and guide the silencing complex to degrade the target miRNA or repress the translation of the target miRNA according to the degree of complementarity. Recent studies have shown that miRNAs are involved in a variety of regulatory pathways, including development, viral defense, hematopoietic processes, organ formation, cell proliferation and apoptosis, fat metab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q1/6841C12Q2525/207C12Q2537/125C12Q2563/107
Inventor 刘苏燕吴诗扬董艳
Owner SUREXAM BIO TECH
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