CN107815495B

CN107815495B - Method for detecting plasma circular RNA marker related to non-small cell lung cancer

Info

Publication number: CN107815495B
Application number: CN201711331148.3A
Authority: CN
Inventors: 杭栋; 戴俊程; 靳光付; 马红霞; 胡志斌; 沈洪兵
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2021-11-12
Anticipated expiration: 2037-12-13
Also published as: CN107815495A

Abstract

The invention discloses a method for detecting a plasma circular RNA marker related to non-small cell lung cancer, which comprises the following steps: step 1, extracting peripheral blood plasma; step 2, extracting plasma RNA; and 3, detecting the circRNA by Real-Time RT PCR. The method can be applied to detecting plasma circular RNA markers related to the non-small cell lung cancer, and has an important auxiliary effect on the diagnosis of the tumor. The nematode circRNA is used as an internal reference for the first time, and the technical bottleneck that the circRNA cannot be quantified well at present is solved.

Description

Method for detecting plasma circular RNA marker related to non-small cell lung cancer

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a method for detecting a plasma circular RNA marker related to non-small cell lung cancer.

Background

Lung cancer is the most globally prevalent malignant tumor at present, and the 5-year survival rate is often lower than 15%. Due to the continuously increasing tobacco consumption and the increasingly aggravated environmental pollution, the lung cancer morbidity and mortality situation in China is more severe, if effective control measures are not taken in time, and 100 thousands of lung cancer patients in China are predicted to become the first lung cancer kingdom in the world by 2025. Research shows that 5-year survival rate of early lung cancer (stage I) patients after operation can reach more than 50%. Therefore, the diagnosis of lung cancer at an early stage and the effective treatment are the key points for prolonging the life cycle and life quality of lung cancer patients.

However, most lung cancer diagnosis methods have different degrees of defects. Pathological examination as a diagnostic gold standard is mostly detection of target tissues obtained by operation or puncture, and has large physical and psychological trauma to patients. However, the conventional tumor markers such as CEA, CA125, CA19-9 and CYFRA21-1 lack sufficient sensitivity and specificity. In addition, the protein serum marker based on the mass spectrometry provides a new exploration direction for early diagnosis of tumors, however, the technology is difficult and high in cost, and the serum protein serving as the marker is often limited by the stability of the serum protein. The circular RNA is a non-coding RNA which is newly discovered internationally, has tissue specificity, expression specificity and good stability, is proved to participate in important physiological and pathological processes of organisms, and is expected to become a new biomarker.

There is a need in the art for a method for detecting plasma circular RNA (circRNA) markers.

Disclosure of Invention

The invention aims to provide a method for detecting a plasma circular RNA marker related to non-small cell lung cancer. The method can be applied to detecting plasma circular RNA markers related to the non-small cell lung cancer, and has an important auxiliary effect on the diagnosis of the tumor. The nematode circRNA is used as an internal reference for the first time, and the technical bottleneck that the circRNA cannot be quantified well at present is solved.

The specific technical scheme is as follows:

a method for detecting a plasma circular RNA marker related to non-small cell lung cancer comprises the following steps:

step 1, peripheral blood plasma extraction: collecting 5ml of morning-start fasting venous blood by adopting a vacuum anticoagulation EDTA blood collection tube for each research object, centrifuging within 12 hours according to a standard method, separating plasma into 1.5ml centrifuge tubes, and preserving two parts at-80 ℃ for later use;

step 2, extracting plasma RNA: 200ul plasma was pretreated by centrifugation at 12000rpm for 10min at 4 ℃. Extracting plasma RNA according to the operational flow of Trizol LS reagent of Invitrogen company and Kit RNeasy Plus Mini Kit of Qiagen company, adding 100ng nematode total RNA as quality control and internal reference standard, purifying the plasma RNA by a centrifugal column, eluting, storing at-80 ℃ for later use, and detecting the purity and concentration of the RNA by a NanoDrop ND-2000 instrument;

step 3, Real-Time RT PCR detection of circRNA: designing a reverse primer specific to the circRNA, carrying out RNA reverse transcription by adopting a reverse transcription kit produced by Takara company, detecting the expression level of the circRNA in plasma by adopting a SYBR-Green dye method, carrying out a reaction system and conditions according to the specification of the kit, carrying out three parallel experiments on each sample, carrying out standardization by taking the circRNA of nematodes as an internal reference, and representing the relative expression value of the circRNA by adopting a delta Ct quantitative method and 2-delta Ct.

Compared with the prior art, the invention has the beneficial effects that:

1) the circRNA has rich content in plasma, stable property and quantitative detection; 2) the detection indexes are minimally invasive and cheap, can be applied to population screening and clinical detection, and are easy to popularize and apply; 3) the invention also provides technical guidance for the research of other disease biomarkers. 4) The invention combines basis, clinic, theory and application, is expected to improve the clinical diagnosis rate of early lung cancer and improve the treatment effect and the life quality of patients.

Drawings

FIG. 1 is a schematic flow chart of the method for detecting plasma circular RNA markers associated with non-small cell lung cancer according to the present invention;

FIG. 2 is the results of the example, wherein FIG. 2A is the dissolution curve obtained by applying Real-Time RT PCR to detect lung cancer related circFARSA, a single peak indicates primer specificity and results are stable; FIG. 2B is a dissolution curve obtained by simultaneously detecting nematode circRNA cel-9 using Real-Time RT PCR, and a single peak indicates that the primers are specific and the result is stable;

FIG. 3 is a graph showing that the relative expression levels of circFARSA in plasma of 50 lung cancer patients and 50 healthy controls were compared using the above experimental conditions, and the results show that the expression levels of circFARSA in lung cancer patients are significantly increased;

figure 4 is a graph reflecting the sensitivity and specificity of using plasma circFARSA to distinguish lung cancer patients from healthy controls, with a 0.71 area under the curve, indicating that this biomarker performs well for identifying lung cancer.

Detailed Description

The technical solutions of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.

As shown in fig. 1:

extracting peripheral blood plasma: each subject collected 5ml of morning fasting venous blood (patient blood specimen before surgery and radiotherapy) with a vacuum anticoagulation (EDTA) blood collection tube, centrifuged within 12 hours according to standard method, separated the plasma into 1.5ml centrifuge tubes (two portions stored), and stored at-80 deg.C for further use.

② extracting plasma RNA: plasma RNA is extracted according to the operational procedures of Trizol LS reagent of Invitrogen company and Kit of Qiagen company (RNeasy Plus Mini Kit), the plasma is pretreated by centrifugation at 12000rpm for 10min at 4 ℃, then 100ng of nematode total RNA is added as a quality control and internal reference standard, the plasma RNA is purified by a centrifugal column, and the RNA is stored at-80 ℃ for standby after elution. The purity and concentration of RNA were determined using a NanoDrop ND-2000 instrument.

③ Real-Time RT PCR detection of circRNA: designing a circRNA specific reverse primer, carrying out RNA reverse transcription by adopting a reverse transcription kit produced by Takara company, detecting the expression level of the circRNA in plasma by adopting a SYBR-Green dye method (Real-time qPCR Master Mix, Takara, Japan), carrying out three parallel experiments on each sample according to the instruction of the kit, and carrying out standardization by taking nematode circRNA as an internal reference. The delta Ct quantification method is adopted, and 2-delta Ct represents the relative expression value of the circRNA.

Example (b): in the case of circFARSA, the specific reverse primer: 5'-GCTCCTTCTGGAACTTTGAC-3', 5'-TTGCTCACCCAGTAGGTCTT-3', respectively;

nematode internal control cel _9 internal control: 5'-TTGCAGCTCTCATAGAAGGAACCG-3', 5'-GTTTCAGCCGAGACTAGACTTTGAGC-3' are provided.

The main results are shown in fig. 2-4.

The Real-Time RT PCR is adopted to detect the internal references of the circFARSA and the nematode internal reference cel _9, the peak value of the dissolution curve is single, and the primer specificity and the result are stable and reliable.

The expression level of circFARSA in the plasma of non-small cell lung cancer (NSCLC) cases is significantly higher than that of a healthy control group (P <0.001), and the expression level can be used as a diagnostic marker.

The area under the receiver operating characteristic curve (ROC) (AUC) of circrsa in plasma was 0.71, indicating that circrsa is more accurate for diagnosing non-small cell lung cancer.

The invention is not limited to the above embodiments, and any simple changes or equivalent substitutions which can be obviously made by those skilled in the art within the technical scope of the invention are within the technical scope of the invention.

Sequence listing

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Claims

1. The application of a primer for detecting nematode circRNA cel _9 as an internal reference primer in the process of preparing a plasma circular RNA marker detection reagent related to non-small cell lung cancer is characterized in that the primer is as follows: 5'-TTGCAGCTCTCATAGAAGGAACCG-3', 5'-GTTTCAGCCGAGACTAGACTTTGAGC-3' are provided.