Method for identifying miRNA in blood serum of colon cancer patient by Solexa technology

A technology of microRNA and patient serum, applied in the field of biomedicine, can solve the problems of low throughput, lack of vigilance in diagnosing colon cancer, sensitivity and repeatability improvement, and achieve high sensitivity effect

Inactive Publication Date: 2008-11-05
NANJING UNIV
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  • Abstract
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Problems solved by technology

[0003] Colon cancer is not easy to diagnose early for the following reasons: 1) The onset is hidden, the clinical manifestations lack specificity, and it is already in the middle and late stage when it is clinically diagnosed
2) Physicians are not vigilant about the connection between symptoms such as abdominal pain, diarrhea, blood in the stool, anemia and colon cancer
3) Lack of vigilance in diagnosing colon cancer in special circumstances, such as colon cancer in young people is more likely to be neglected, and colon cancer in young people is often poorly differentiated and has a poor prognosis, thus delaying diagnosis
RT-PCR and Real-time PCR have problems such as high experimental cost and low throughput
Northern blotting can visually display and detect precursor and mature microRNAs, but the operation is cumbersome, the sensitivity and the experimental throughput are low
Although biochip technology has high throughput and low cost, sensitivity and repeatability need to be improved

Method used

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  • Method for identifying miRNA in blood serum of colon cancer patient by Solexa technology
  • Method for identifying miRNA in blood serum of colon cancer patient by Solexa technology
  • Method for identifying miRNA in blood serum of colon cancer patient by Solexa technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Solexa sequencing technology was used to sequence the serum samples of patients with colon cancer and analyze the expression levels of each contained microRNA. The specific steps are:

[0041] (1) Collect serum samples from patients with colon cancer.

[0042] (2) Trizol method (Invitrogen Company) extracted total RNA from 100ml of mixed serum of the above patients, concentrated RNA by isopropanol precipitation, and quantified it with a spectrophotometer (100ml serum can usually enrich about 10-20μg of total RNA).

[0043] (3) About 10-20 μg of serum total RNA from patients with colon cancer was used to detect and analyze RNA samples using Solexa sequencing technology. The specific plan is:

[0044] a. Apply PAGE electrophoresis to purify and obtain RNA molecules with a length less than 30nt. An adapter is added to both ends of the RNA molecule.

[0045] b. While using the Solexa chip to immobilize one end of the RNA adapter molecule, since the surface of ...

Embodiment 2

[0066] Example 2 Changes of microRNA in serum of patients with colon cancer compared with normal physiological state

[0067] Normal human serum microRNA was sequenced by Solexa technology, and the results are shown in Table 3.

[0068] Table 3 Solexa sequencing results of normal human serum

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[0070]

[0071]

[0072]

[0073]

[0074]

[0075] If the value of Solexa sequencing is less than 1 copy number, we consider it unreliable and default to zero after filtering. There are 222 microRNAs in the filtered colon cancer patient serum (see Table 2). There are 167 microRNAs (see Table 3) in the filtered normal human serum. We believe that when the change factor of the microRNA is greater than 5, and the difference in absolute value is greater than 20 copy numbers, then the microRNA is significantly changed, otherwise it is regarded as unchanged. According to this rule, we compared the microRNAs in the serum of patients with colon cancer and the serum...

Embodiment 3

[0076] Embodiment 3 is used for the making of the serum microRNA test kit of diagnosing colon cancer

[0077] The production process and operation process of the microRNA kit for diagnosing and predicting colon cancer are based on Solexa sequencing technology, combined with quantitative and semi-quantitative PCR technology.

[0078] Through the Solexa sequencing method, the serum microRNA expression profiles of normal people and colon cancer patients were first determined, and then a series of serum microRNAs with large expression levels and differences in colon cancer and normal physiological conditions were screened to predict whether colon cancer occurred. Cancer and diagnostic indicators of disease severity. Table 4 shows that compared with normal human serum, the significantly changed microRNAs in the serum of colon cancer patients can screen out a batch of serum microRNAs as molecular markers for the diagnosis of colon cancer.

[0079] Table 4 colon cancer patient's ser...

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Abstract

The invention discloses a method for identifying micro ribonucleic acid in the blood serum of colon carcinoma patients through a Solexa technique, belongs to the technical field of biological medicine, and particularly relates to the Solexa technique that researches on the micro ribonucleic acid expression in the blood serum and the application of the Solexa technique in colon carcinoma diagnosis. The method for identifying the micro ribonucleic acid in the blood serum of colon carcinoma patients comprises measuring an expression profiling of the micro ribonucleic acid in the blood serum of a colon carcinoma subject by applying the Solexa sequencing method. The Solexa sequencing method has the advantages of wide measuring pedigree, high sensitivity, convenient materials obtaining and easy sample storing, etc., and can be widely used for relevant tasks such as disease census measuring, which can improve low specificity and low sensitivity that are caused by individual difference and are difficult to be overcome by single markers, and remarkably improve the clinical relevance ratio of diseases, and is an effective method for disease diagnosis in early stages.

Description

1. Technical field [0001] The invention belongs to the technical field of biomedicine. It specifically relates to a Solexa technology for measuring the expression of microRNA in serum, and the application of the Solexa technology in the clinical diagnosis of colon cancer. 2. Background technology [0002] In recent years, the incidence of colon cancer has been on the rise, ranking fourth among malignant tumors. The pathogenesis of colon cancer is not yet fully understood, and it is believed that its occurrence is mainly the result of long-term interaction between genetic and environmental factors. Studies have shown that it can take up to 10 years for colon cancer to develop and around five years for the progression from an endoscopically identifiable adenoma alone to an invasive carcinoma. Early colon cancer can be cured, but the prognosis of advanced colon cancer is poor. At present, the 5-year survival rate of colon cancer patients in China is only about 60%. Early det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈江宁蔡星殷媛张峻峰张辰宇王可慧郭继刚陈熹王进曾柯
Owner NANJING UNIV
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