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Pancreatic cancer marker, and detection method, kit and biochip thereof

A marker, pancreatic cancer technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc.

Active Publication Date: 2014-09-17
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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  • Pancreatic cancer marker, and detection method, kit and biochip thereof
  • Pancreatic cancer marker, and detection method, kit and biochip thereof
  • Pancreatic cancer marker, and detection method, kit and biochip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 RT-PCR Experiment of MicroRNA in Serum / Plasma

[0070] 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:

[0071] (1) Collect serum / plasma from mice, rats, normal people and certain patients;

[0072] (2) Preparation of cDNA samples. There are two schemes for this operation. One scheme is to directly perform reverse transcription reaction on 10 μl serum / plasma, and the other is to use Trizol reagent (Invitrogen) to extract serum / plasma total RNA first (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 mM each 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 incubation at...

Embodiment 2

[0076] Example 2 real-time PCR experiment of microRNA in serum / plasma

[0077] In order to study the specific changes of serum / plasma microRNA in the course of pancreatic 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 amount of each microRNA relative to the standard internal reference can be expressed by Equation 2 -ΔCT Indicates that ΔCT=CT sample-CT internal reference. The patient's serum / plasma sample and normal human serum / ...

Embodiment 3

[0079] Example 3 Serum / Plasma MicroRNA Chips for Diagnosis of Pancreatic Cancer

[0080] The chip operation process is as follows:

[0081] (1) extract total RNA in serum / plasma, and formaldehyde denaturing gel electrophoresis to detect the quality of total RNA;

[0082] (2) Isolation of microRNA: Take 50-100 μg of total RNA and use Ambion's miRNAIsolation Kit (Cat#.1560) to isolate microRNA;

[0083] (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;

[0084] (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 after dryi...

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Abstract

The invention provides a pancreatic cancer marker, and a detection method, a kit and a biochip thereof. The pancreatic cancer marker provided by the invention contains 36 kinds of micro ribonucleic acids which stably exist in the serum / plasma of a person receiving the test and can be detected. The invention also provides a kit and a biochip of tools or elements for detecting the pancreatic cancer marker. The combination, the method, the kit and the biochip provided by the invention can be used for auxiliary diagnosis and differential diagnosis of pancreatic cancers, predication of occurrence and recrudescence of disease complications, evaluation of the curative effect, screening of active ingredients of medicaments, evaluation of pesticide effectiveness and the like, and has the advantages of wide detection range, high sensitivity, low detection cost, readily available raw materials, easy storage of samples and the like; and the method is widely applied to work related to the general survey of pancreatic cancers, improves the specificity and sensitivity which are low in the single marker due to individual difference, obviously increases clinical detection rate of pancreatic cancers and becomes an effective method for early diagnosis of pancreatic cancers.

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, and also relates to various clinical indications of pancreatic cancer. Specifically, the present invention is a method for detecting microRNA in serum / plasma of patients with pancreatic cancer, through the change of microRNA in serum / plasma of patients with pancreatic cancer, in vitro diagnosis of pancreatic cancer and chronic pancreatitis, and judgment of pancreatic cancer The pathogenesis process predicts the occurrence of pancreatic cancer complications, the probability of pancreatic cancer recurrence, and the prognosis of pancreatic cancer, and analyzes the efficacy and efficacy of drugs. Background technique [0002] Pancreatic cancer is a tumor with a very high mortality rate (~99.9%, after diagnosis). U.S. Incidence: Estimated 32,180 new cases in 2005, accounting for ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 张辰宇刘锐王成巴一张春妮曾科
Owner JIANGSU MICROMEDMARK BIOTECH
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