Marker for detecting colon and rectum cancer as well as detection method, kit and biological chip thereof

A technology for colorectal cancer and detection markers, applied in biochemical equipment and methods, microbiological determination/testing, 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

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Problems solved by technology

However, the wide application of this method in clinical pra...

Method used

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  • Marker for detecting colon and rectum cancer as well as detection method, kit and biological chip thereof
  • Marker for detecting colon and rectum cancer as well as detection method, kit and biological chip thereof
  • Marker for detecting colon and rectum cancer as well as detection method, kit and biological chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The RT-PCR experiment of microRNA in embodiment 1 serum / plasma

[0082] 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:

[0083] (1) Collect serum / plasma from mice, rats, normal people and certain patients;

[0084] (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

[0090] The real-time PCR experiment of microRNA in embodiment 2 serum / plasma

[0091] In order to study the specific changes of serum / plasma microRNA in colorectal cancer disease process, 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 sample ar...

Embodiment 3

[0093] Embodiment 3 is used for diagnosing the serum / plasma microRNA chip of colorectal cancer

[0094] The chip operation process is as follows:

[0095] (1) extract total RNA in serum / plasma, and formaldehyde denaturing gel electrophoresis to detect the quality of total RNA;

[0096] (2) Isolation of microRNA: Take 50-100 μg of total RNA and use Ambion's miRNA Isolation Kit (Cat#.1560) to isolate microRNA;

[0097] (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;

[0098] (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 scann...

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Abstract

The invention relates to a marker for detecting colon and rectum cancer by utilizing 86 specific ribonucleic acids stably existing in the serum/plasma of a human body, as well as a method, a relevant kit and a biological chip for detecting the marker. The method can be used in the aspects of diagnosing and differentially diagnosing the colon and rectum cancer and predicting the generation and the recurrence of the complications of diseases, evaluating the curative effect, screening the active ingredients in drugs, evaluating the drug effect and the like, and has the advantages of wide detection pedigree, high sensitivity and low detection cost, material is convenient for obtainment, samples are easy to store and the like. The method can be widely used for generally surveying the colon and rectum cancer and relevant work and improving low specificity and low sensitivity which are brought by the individual difference difficultly and are difficult to overcome by the single marker, thereby obviously enhancing the clinical detection rate of the colon and rectum cancer and becoming an effective means for early diagnosing the colon and rectum cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the separation, qualitative and quantitative analysis of microRNA molecules in human serum / plasma, and also relates to various clinical indications of colorectal cancer. Specifically, the present invention is a method for detecting microRNA in serum / plasma of patients with colorectal cancer, through the change of microRNA in serum / plasma of patients with colorectal cancer, in vitro diagnosis of colorectal cancer, and judgment of colorectal cancer The pathogenesis process predicts the occurrence of colorectal cancer complications, the probability of colorectal cancer recurrence, and the prognosis of colorectal cancer, and analyzes the efficacy and efficacy of drugs. Background technique [0002] Colorectal cancer is a common malignant tumor of the digestive tract, and its incidence rate is second only to gastric cancer and esophageal cancer. Among the common malignant tumor deaths in m...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张辰宇曾科李海进
Owner JIANGSU MICROMEDMARK BIOTECH
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