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Breast cancer detecting marker as well as detecting method, kit and biological chip thereof

A technology for detection of markers and biochips, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of wide application limitations and difficulty in obtaining materials

Inactive Publication Date: 2011-03-23
JIANGSU MICROMEDMARK BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the wide application of this method in clinical practice is limited due to the difficulty in obtaining tissue samples.

Method used

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  • Breast cancer detecting marker as well as detecting method, kit and biological chip thereof
  • Breast cancer detecting marker as well as detecting method, kit and biological chip thereof
  • Breast cancer detecting marker as well as detecting method, kit and biological chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] The RT-PCR experiment of microRNA in embodiment 1 serum / plasma

[0080] Using RT-PCR technology to discover and prove that various microRNAs exist stably in human and animal serum / plasma, and their expression is quite abundant. The specific steps are:

[0081] (1) Collect serum / plasma from mice, rats, normal people and certain patients;

[0082] (2) Preparation of cDNA samples. There are two schemes for this operation, one scheme is to directly carry out reverse transcription reaction on 10 μl serum / plasma, and the other is to use Trizol reagent (Invitrogen Company) to first extract serum / plasma total RNA (10ml serum / plasma can usually enrich about 10 μg Left and right RNA), and then cDNA was obtained by RNA reverse transcription reaction. The reverse transcription reaction system included 4 μl 5×AMV buffer, 2 μl 10 mMeach dNTP mixture (Takara), 0.5 μl RNase Inhibitor (Takara), 2 μl AMV (Takara) and 1.5 μl gene-specific reverse primer mixture. The reaction steps are...

Embodiment 2

[0088] The real-time PCR experiment of microRNA in embodiment 2 serum / plasma

[0089] In order to study the specific changes of serum / plasma microRNA in the course of breast cancer disease, the quantitative PCR experiment of serum / plasma microRNA was carried out. The experimental principle and experimental steps of quantitative PCR are the same as RT-PCR, the only difference is that the fluorescent dye EVA GREEN is added during PCR. The instrument used was ABI Prism 7300 fluorescence quantitative PCR instrument, and the reaction conditions were 95°C, 5 minutes for 1 cycle → 95°C, 15 seconds, 60°C, 1 minute for 40 cycles. The data processing method is the ΔΔCT method, and CT is set as the cycle number when the reaction reaches the threshold value, then the expression level of each microRNA relative to the standard internal reference can be expressed by equation 2-ΔCT, where ΔCT=CT sample-CT internal reference. The patient's serum / plasma sample and normal human serum / plasma sam...

Embodiment 3

[0091] Embodiment 3 is used for the serum / plasma microRNA chip of diagnosing breast cancer

[0092] The chip operation process is as follows:

[0093] (1) extract total RNA in serum / plasma, and formaldehyde denaturing gel electrophoresis to detect the quality of total RNA;

[0094] (2) Isolation of microRNA: Take 50-100 μg of total RNA and use Ambion's miRNA Isolation Kit (Cat#.1560) to isolate microRNA;

[0095] (3) Fluorescence labeling of microRNA samples: use T4 RNA ligase labeling method for fluorescent labeling, and then precipitate with absolute ethanol, dry and use for chip hybridization;

[0096] (4) Hybridization and washing: Dissolve RNA in 16 μL of hybridization solution (15% formamide; 0.2% SDS; 3×SSC; 50×Denhardt’s solution), and hybridize overnight at 42°C. After the hybridization, wash in a liquid containing 0.2% SDS and 2×SSC at about 42°C for 4 minutes, then wash in a 0.2×SSC liquid for 4 minutes at room temperature, and the slides can be used for scanning ...

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Abstract

The invention relates to a breast cancer detecting marker by utilizing 16 specific tiny ribonucleic acids steadily existing in blood serum and blood plasma of a human body as well as a detecting method, a kit and a biological chip thereof, and the detecting marker can be used in the aspects of diagnosis and differential diagnosis of breast cancer, occurrence and recurrence forecast as well as efficacy assessment of disease complications, screening and therapeutic evaluation of medicinal active ingredients and the like, and has the advantages of wide detection pedigree, high sensitivity and low detection cost, the materials are conveniently taken, the samples are easy to store and the like. The method can be widely used in related work such as breast cancer census, the defects of low specificity and low sensitivity caused by insuperable individual difference of a single marker can be improved and the clinical detectable rate of the breast cancer is notably improved, and is an effective mean of early breast cancer diagnosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the separation, qualitative and quantitative analysis of microribonucleic acid molecules in human serum / plasma, as well as various clinical indications of breast cancer. Specifically, the present invention is a specific microRNA that stably exists in the serum / plasma of breast cancer patients as a detection marker for breast cancer and a method for detecting the marker, related kits and biochips. The change of microRNA in serum / plasma can diagnose breast cancer in vitro, judge the pathogenesis of breast cancer, predict the occurrence of breast cancer complications, the probability of breast cancer recurrence, and the prognosis of breast cancer, and analyze the efficacy and efficacy of the drug. Background technique [0002] Breast cancer has become the malignant tumor with the highest incidence rate among women in the world. About 1.2 million women in the world suffer from breast cance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 张辰宇曾科李海进
Owner JIANGSU MICROMEDMARK BIOTECH
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