Gastric cancer detection marker and detecting method thereof, kit and biochip

A technology for detecting markers and biochips, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc.

Active 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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  • Gastric cancer detection marker and detecting method thereof, kit and biochip
  • Gastric cancer detection marker and detecting method thereof, kit and biochip
  • Gastric cancer detection marker and detecting method thereof, kit and biochip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] The RT-PCR experiment of microRNA in embodiment 1 serum / plasma

[0084] 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:

[0085] (1) Collect serum / plasma from mice, rats, normal people and certain patients;

[0086] (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

[0092] The real-time PCR experiment of microRNA in embodiment 2 serum / plasma

[0093] In order to study the specific changes of serum / plasma microRNA in the disease process of gastric cancer, 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 Prism7300 fluorescent 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

[0095] Embodiment 3 is used for diagnosing the serum / plasma microRNA chip of gastric cancer

[0096] The chip operation process is as follows:

[0097] (1) extract total RNA in serum / plasma, and formaldehyde denaturing gel electrophoresis to detect the quality of total RNA;

[0098] (2) Isolation of microRNA: Take 50-100 μg of total RNA and use Ambion's miRNA Isolation Kit (Cat#.1560) to isolate microRNA;

[0099] (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;

[0100] (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 gastric cancer detection marker, and a detecting method thereof, a related kit and a biochip. The gastric cancer detection marker comprises 21 specific micro RNAs stable in human serum / plasma. The marker which is broad in detection spectrum, high in sensitivity, low in detection cost, easy to be got and stored can be used for diagnosing and differentially diagnosing the gastric cancer, predicating occurrence and reoccurrence of diseases and conditions, evaluating therapeutic efficiency and efficacy and screening active pharmaceutical ingredients and the like. The method can be widely applied to cancer survey and related work, improves low specificity and low sensitivity caused by individual difference which is difficult for a single marker to overcome, and remarkably improves the clinical detection rate of the gastric cancer. Therefore, the method is an effective means to diagnose early breast cancer.

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 gastric cancer. Specifically, the present invention is a specific microRNA that stably exists in the serum / plasma of patients with gastric cancer as a detection marker for gastric cancer and a method for detecting the marker, related kits and biochips. Changes in microRNA, in vitro diagnosis of gastric cancer and chronic pancreatitis, judgment of gastric cancer pathogenesis, prediction of gastric cancer complications, probability of gastric cancer recurrence, and prognosis of gastric cancer, and analysis of drug efficacy and efficacy. Background technique [0002] Gastric cancer is one of the common malignant tumors in my country, and its incidence rate ranks first among all kinds of tumors in China. Adenocarcinoma accounts for 95% ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 张辰宇曾科李海进巴一刘锐
Owner JIANGSU MICROMEDMARK BIOTECH
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