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Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof

Active Publication Date: 2013-01-16
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low expression of miRNA in serum / plasma, finding a detection method with high sensitivity, easy operation and low cost is an urgent problem to be solved in the clinical detection of serum / plasma miRNA.
On the other hand, using only one serum / plasma miRNA as a tumor marker is often not specific enough. If multiple miRNAs are used in combination and combined with other types of tumor marker detection, it is expected to significantly improve the accuracy of diagnosis
[0006] However, there are no reports of relatively stable biomarkers for the diagnosis of pancreatic cancer. If abnormally expressed serum / plasma miRNAs can be screened out as biomarkers and corresponding diagnostic kits can be developed, it will be beneficial for pancreatic cancer in my country. The status quo of diagnosis will be a powerful push

Method used

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  • Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof
  • Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof
  • Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Collection of samples and arrangement of sample data

[0071] The cases are new cases of pancreatic cancer collected at Huai'an Cancer Hospital and Jiangsu Cancer Hospital from June 2007 to December 2010, all of which were confirmed by histopathology. Controls were healthy individuals who had been screened for community diseases during the same period, and were frequency-matched with cases by gender and age (±5 years). The samples used for the research were collected at the same time, and the conditions of sampling, aliquoting, and storage were uniform. After sorting out the sample data, the inventor selected 48 samples that met the following standards as the TLDA chip detection and subsequent series of qRT-PCR verification Experimental sample:

[0072] 1. New cases of pancreatic cancer

[0073] 2. No surgery, radiotherapy and chemotherapy before blood collection, no preoperative radiotherapy and chemotherapy

[0074] 3. Control of healthy people matching the age an...

Embodiment 2

[0076] Example 2 TLDA chip detection of miRNA in serum / plasma

[0077] The 24 pancreatic cancer patients and 24 healthy controls were tested by TLDA chip to obtain relevant results. The specific steps are:

[0078] 1. Take 600μl of serum from patients in the "pancreatic cancer case" group and the "healthy control" group respectively, and add 3 times the volume of Trizol reagent;

[0079] 2. Phase separation: place at room temperature for 15 minutes, add the final concentration to 10 -4 pmol / μl cel-39 (TAKARA) was used as the internal control, and then added the same volume of chloroform as the plasma, shaking for 50s, room temperature for 15min, 14,000rpm, 4℃, centrifugation for 15min;

[0080] 3. RNA precipitation: transfer the water phase to a new 15ml centrifuge tube, add 1.5 times the volume of the water phase absolute ethanol, and mix well;

[0081] 4. Use QIAGEN miRNeasy kit to enrich RNA: Pipette 700μl sample into the spin column each time, centrifuge at 14,000rpm for 15s, disca...

Embodiment 3

[0087] Example 3 qRT-PCR experiment of miRNA in serum / plasma

[0088] According to the above TLDA results, select miRNAs that meet the following conditions to further verify with qRT-PCR method: 1) The CT values ​​of the two groups of subjects in the TLDA chip are not greater than 35 to improve the detection efficiency; 2) The ΔΔCT in the TLDA chip is greater than 2. Design primers for reverse transcription and qRT-PCR for the selected miR-451 and miR-409-3p miRNAs (see Table 1). The qRT-PCR detection of miRNA was performed on individual serum individuals in the "pancreatic cancer case" group and the "healthy control" group. Strict quality control was implemented throughout the research process. Each sample was tested three times in a row. All tests are blinded, that is, done without knowing the background of the sample to avoid bias. Two methods of dye method and probe method were used for qRT-PCR detection.

[0089] Table 1 Related miRNA primer information

[0090]

[0091] (1...

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Abstract

The invention belongs to the fields of genetic engineering and oncology, and discloses a blood serum / blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof. The marker is formed by combining miR-451 and miR-490-3p. The marker and primers of the marker can be used for preparing a diagnostic kit and are used for the auxiliary diagnosis of pancreatic cancer.

Description

Invention field [0001] The invention belongs to the field of genetic engineering and oncology, and relates to a serum / plasma miRNA marker related to pancreatic cancer and its application. Background technique [0002] Pancreatic cancer is one of the common gastrointestinal tumors. It is estimated that there were 270,000 new cases of pancreatic cancer worldwide in 2008. In my country, the incidence of pancreatic cancer has increased by nearly 6 times in the past 20 years, especially in economically developed areas, the incidence of pancreatic cancer has been the same as that of Western countries. Pancreatic cancer has a high degree of malignancy and a very poor prognosis. Due to its special biological behavior, that is, it is easy to invade blood vessels and nerves. The tumor metastasizes at a very early age. It is not sensitive to conventional chemotherapy, radiotherapy and immunotherapy. The 5-year survival rate is only about 5%. In recent years, as the incidence and mortality...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12Q1/68
Inventor 潘峰高勇祁付珍胡志斌董静闻洋
Owner NANJING MEDICAL UNIV
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