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Cervical cancer detection markers and detection method, kit and biochip thereof

A technology for detecting markers and biochips, which is applied in various clinical indication fields of cervical cancer, and can solve problems such as difficulty in obtaining materials and limitations in wide application

Inactive 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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  • Cervical cancer detection markers and detection method, kit and biochip thereof
  • Cervical cancer detection markers and detection method, kit and biochip thereof
  • Cervical cancer detection markers and detection method, kit and biochip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The RT-PCR experiment of microRNA in embodiment 1 serum / plasma

[0077] 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:

[0078] (1) Collect serum / plasma from mice, rats, normal people and certain patients;

[0079] (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

[0085] The real-time PCR experiment of microRNA in embodiment 2 serum / plasma

[0086] In order to study the specific changes of serum / plasma microRNA in the course of cervical 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 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

[0088] Embodiment 3 is used for the serum / plasma microRNA chip of diagnosing cervical cancer

[0089] The chip operation process is as follows:

[0090] (1) extract total RNA in serum / plasma, and formaldehyde denaturing gel electrophoresis to detect the quality of total RNA;

[0091] (2) Isolation of microRNA: Take 50-100 μg of total RNA and use Ambion's miRNA Isolation Kit (Cat#.1560) to isolate microRNA;

[0092] (3) Fluorescence labeling of microRNA samples: fluorescent labeling by T4RNA ligase labeling method, and then precipitated with absolute ethanol, dried and used for chip hybridization;

[0093] (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 17 specific micro ribonucleic acids serving as cervical cancer detection markers, existing in human serum / plasma and capable of being detected, and a method, a related kit and a biochip for detecting the markers. The markers can be used for diagnosis and identification of cervical cancer, prediction of occurrence and relapse of disease complications, curative effect evaluation, screening of medicinal active ingredients, medicinal effect evaluation and the like, have the advantages of broad detection spectrum, high sensitivity, low detection cost, convenience for taking materials and the like, and are easy to store samples. The method can be widely used for related operations such as cervical cancer general investigation and the like, improves low specificity and low sensitivity caused by individual difference difficult to be overcome by single marker, remarkably improves the clinical detection rate of the cervical cancer, and becomes an effective means for early diagnosing the cervical 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, as well as various clinical indications of cervical cancer. Specifically, the present invention is a specific microRNA that stably exists in the serum / plasma of patients with cervical cancer as a detection marker for cervical cancer and a method for detecting the marker, related kits and biochips. The change of microRNA in serum / plasma can be used to diagnose cervical cancer in vitro, judge the pathogenesis of cervical cancer, predict the occurrence of cervical cancer complications, the probability of cervical cancer recurrence, and the prognosis of cervical cancer, and analyze the efficacy and curative effect. Background technique [0002] Cervical cancer is one of the most common malignant tumors. In my country, the occurrence of cervical cancer accounts for the first place among female...

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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