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Non-small cell lung cancer marker as well as detection method and application thereof

A technology for non-small cell lung cancer and detection methods, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. question

Active Publication Date: 2015-06-10
JIANGSU MICROMEDMARK BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application needs to use many types of markers, the corresponding probe library is relatively large, and the accuracy rate is not ideal

Method used

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  • Non-small cell lung cancer marker as well as detection method and application thereof
  • Non-small cell lung cancer marker as well as detection method and application thereof
  • Non-small cell lung cancer marker as well as detection method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] The RT-PCR experiment of microRNA in embodiment 1 serum / plasma

[0091] 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:

[0092] (1) Collect serum / plasma from mice, rats, normal people and certain patients;

[0093] (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 mix. The reaction steps are incubation...

Embodiment 2

[0097] The real-time PCR experiment of microRNA in embodiment 2 serum / plasma

[0098] In order to study the specific changes of serum / plasma microRNA in the course of non-small cell lung 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 / plasm...

Embodiment 3

[0100] Embodiment 3 is used for diagnosing the serum / plasma microRNA chip of non-small cell lung cancer

[0101] The chip operation process is as follows:

[0102] (1) Extract total RNA from serum / plasma, and detect the quality of total RNA by formaldehyde denaturing gel electrophoresis;

[0103] (2) Isolation of microRNA: Take 50-100 μg of total RNA and use Ambion's miRNA Isolation Kit (Cat#.1560) to isolate microRNA;

[0104] (3) Fluorescent labeling of microRNA samples: use T4RNA ligase labeling method for fluorescent labeling, then precipitate with absolute ethanol, dry and use for chip hybridization;

[0105] (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 fo...

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Abstract

The invention provides a non-small cell lung cancer marker and an application thereof. The non-small cell lung cancer marker provided by the invention comprises two or more than two of micro RNA mature bodies, which are stable in human serum / blood plasma and can be detected, such as miR-7, miR-25, miR-193a-3p and miR-483-5p. The invention also provides a probe combination, a kit and a biological chip which are used for detecting the non-small cell lung cancer marker. Besides, the invention also provides a method for detecting micro RNA in human serum of a lung cancer patient. By virtue of change of the micro RNA in the human serum of the lung cancer patient, non-small cell lung cancer is diagnosed in vitro, a non-small cell lung cancer pathogenetic process is predicted, occurrence of complications, non-small cell lung cancer recurrence probability and prognosis of the non-small cell lung cancer are judged, and pharmacological function and curative effect are analyzed.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a non-small cell lung cancer marker and its application, in particular to the use of at least two mature microRNAs selected from four types that are stable and detectable in human serum / plasma as non-small cell lung cancer markers. Small cell lung cancer markers, further, the present invention also provides probe combinations, kits and biochips for detecting the non-small cell lung cancer markers and applications of the non-small cell lung cancer markers, specifically, By detecting the expression levels and changes of the markers in the subject's serum, the in vitro auxiliary diagnosis of non-small cell lung cancer, the prediction of complications, the probability of cancer recurrence, and the prognosis of non-small cell lung cancer can be achieved. Background technique [0002] Lung cancer is the cancer with the highest morbidity and mortality worldwide. The incidence of lung cancer in C...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 张辰宇曾科张春妮陈熹王成
Owner JIANGSU MICROMEDMARK BIOTECH
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