CN102424858A

CN102424858A - Quantitative detection method of serum miRNA-483-5p

Info

Publication number: CN102424858A
Application number: CN2011104585881A
Authority: CN
Inventors: 王效民; 刘平果; 张舟径
Original assignee: Zhongshan Hospital Xiamen University
Current assignee: Zhongshan Hospital Xiamen University
Priority date: 2011-12-31
Filing date: 2011-12-31
Publication date: 2012-04-25

Abstract

A quantitative detection method of serum miRNA-483-5p relates to a detection method of serum. The serum to be detected is mixed with a lysis buffer. RNA is precipitated by a phenol-chloroform method after lysis of the serum and small fragment RNA is reclaimed. The purified miRNA is transformed into cDNA under the catalysis of MMLV enzyme by the use of specific primers. Then cDNA undergoes quantitative PCR detection under the catalysis of EXtaq enzyme by the use of miRNA-483-5p PCR primers and a probe. FAM fluorescence value is detected at 70 DEG C per each cycle. Data is analyzed and compared with a standard sample so as to obtain a quantitative value when cycles are finished. The method provided by the invention performs systemic and complete verifications on the design of the primers and the probe, the use of various catalyzing enzymes and the quantitative method of external parameter, solves the inaccurate quantification problem due to the internal parameter instability in the serum. The method is expected to be applied in clinic and plays a guiding role in the popular research of taking miRNA as a biological tag of various diseases in the serum from now on.

Description

The quantitative detecting method of serum miRNA-483-5p

Technical field

The present invention relates to a kind of detection method of serum, especially relate to the quantitative detecting method of a kind of serum miRNA-483-5p.

Background technology

Chinese patent CN101793882A discloses the proteic mass spectrum screening method of tumor marker Bicr6 in a kind of human serum, and its concrete steps are: get human serum, obtain enrichment with magnetic bead liquid with magnetic bead absorption back wash-out; Enrichment with magnetic bead liquid is added matrix solution, carry out the ground substance assistant laser ionization time of flight mass spectrometry that dissociates by following condition and detect: first ion source voltage: the 20kV; Second ion source voltage: the 18.6kV; Crystal voltage: 7.6kV; The pulse ion time: 320ns; Polarity: just; Matrix suppression mode: gate; Gate intensity: maximum; Suppress high limit: 600Da; Detector: 1.4～1.5kV; Sample rate: 1.00Gs/s; Electronics obtains: strengthen 100mV; Smooth in real time: height; Laser frequency: 20Hz; Laser attenuation: close 73%, scope 20%; Mass-to-charge ratio is that the peak at 4964 ± 1 places is a Bicr6 albumen.This invention has characteristics such as time weak point, sensitivity height.

Summary of the invention

The object of the present invention is to provide the quantitative detecting method of a kind of serum miRNA-483-5p.

The present invention includes following steps:

1) gets serum to be detected and mix, separate out RNA and reclaim small fragment RNA with phenol chlorine method after the cracking with lysate;

2) getting purifying miRNA uses specific primer miRNA is become cDNA under the MMLV enzyme catalysis; PCR primer and the probe of then cDNA being used miRNA-483-5p under the EXtaq enzyme catalysis carry out quantitative PCR detection; Condition is to carry out 95 ℃ of 10s of 40 circulations, 60 ℃ of 10s, 70 ℃ of 10s behind 95 ℃ of 10min; Detect the FAM fluorescent value when whenever circulating in 70 ℃, accomplish circulation post analysis data and standard substance and relatively draw quantitative values; PCR primer and the probe of said miRNA-483-5p are as shown in table 1.

Table 1

In step 1), said serum to be detected can be 400 μ l, the consumption of said lysate can with the same volume of serum to be detected.

In step 2) in, the said quantitative PCR detection of carrying out can adopt ABI7300 quantitative PCR appearance.

The present invention has set up a complete set of micro-miRNA detection by quantitative system, comprises that the selection of miRNA and the expression of key enzyme are synthesized, joined to synthetic, the primer design of standard substance outward.

Compare with existing similar approach, outstanding advantage of the present invention and outstanding effect are following:

So far; About extraction and the detection method of serum miRNA rarely has report; Verified to system complete of the present invention ginseng quantivative approach outside the using of primer, probe design, various katalaze(enzyme)s; Solved that the confidential reference items instability causes quantitative inaccurate drawback in the serum, be expected to this invention is applied to clinical, and miRNA in the serum from now on played guiding function as this popular research of biomarker of various diseases.

Description of drawings

Fig. 1 is miRNA real-time fluorescence quantitative PCR standard substance curve (correction that the adds reference that Ath-miR uses when detecting for the back).In Fig. 1, X-coordinate is miRNA standard substance add-on (having got 10 logarithm), and ordinate zou is a nucleic acid increased logarithmic phase cycle number; Mark ◆ be miR-500a, ■ are miR-483, ▲ be Ath-miR-156; The quantitative formula of Ath-miR-156: y=-3.514x+38.65, R ²=0.993; The quantitative formula of miR-483-5p: y=-3.533x+40.71, R ²=0.997; The quantitative formula of miR-500a: y=-3.265x+40.48, R ²=0.994.

Fig. 2 miRNA-483-5p serum mixes the slurry experimental result.In Fig. 2, ordinate zou is a copies/ml.

Fig. 3 is the expression situation (n represent every group patient number) of serum miRNA-483-5p in hepatitis B dependency liver cancer, chronic hepatitis B, healthy population.In Fig. 3, ordinate zou is serum miR-483-5p amount (log ₁₀Copy/ml).

Embodiment

At first use the serum miRNA high flux screening of miRNA chip technology to the hepatitis B patient of hepatitis B dependency liver cancer and age, sex paired non-liver cancer; Screening utilizes the quantitative PCR detection system of setting up and verifies serum miR-483-5p at the miRNAs at hepatitis B dependency liver cance high-expression.

Standard substance curve experimental technique can entrust the synthetic miRNA standard substance of nucleic acid Synesis Company (can prevent the cutting of RNA excision enzyme through special chemically modified with reference to the known array of http://www.mirbase.org/ DB; The empirical tests ambient stable is placed February and is not degraded), calculate copy number and on demand concentration dilute as outer ginseng.Use the standard substance formation of concentration known, get serum 400 μ l to be detected, and mixes with the volume lysate, quantitative RT-PCR method has been verified the good linear and the highly sensitive (referring to Fig. 1) of detection.

After detection architecture is confirmed again comprehensive literature several miRNA carried out serum mixed slurry checking (referring to Fig. 2); The purpose in this step is to find expression amount miRAN relatively preferably in our detection level can reach; Even have relative expression quantity to change and abandon some; But measure the atomic operated miRNA that is difficult for, the result is as shown in Figure 2, in the miRNA of all selections, has only miR-500a and miR-483-5p that higher expression is arranged.

Serum mixes the slurry experimental technique: get serum 85 μ l concussion mixing; Getting mixing back serum 400 μ l detects according to preceding method; Detect and do standard control simultaneously, calculating formula directly draws the copy number that each group is mixed every kind of miRNA in the slurry through typical curve, gets rid of whereby and expresses low or insignificant miRNA.

Utilize this detection architecture to detect the expression of candidate serum miRNA-483-5p in hepatitis B dependency liver cancer (n=112), chronic viral hepatitis B (n=85), healthy population (n=56); Find miR-483-5p to diagnosing cancer of liver efficient (73%), unite the clinical diagnosis efficiency (sensitivity=83% specific degree=81%) that at present improves liver cancer with the Serum AFP diagnosis index.Therefore, serum miR-483-5p is to hepatitis B dependency diagnosing cancer of liver valuable (referring to Fig. 3).

Claims

1. the quantitative detecting method of serum miRNA-483-5p is characterized in that may further comprise the steps:

2) getting purifying miRNA uses specific primer miRNA is become cDNA under the MMLV enzyme catalysis; PCR primer and the probe of then cDNA being used miRNA-483-5p under the EXtaq enzyme catalysis carry out quantitative PCR detection; Condition is to carry out 95 ℃ of 10s of 40 circulations, 60 ℃ of 10s, 70 ℃ of 10s behind 95 ℃ of 10min; Detect the FAM fluorescent value when whenever circulating in 70 ℃, accomplish circulation post analysis data and standard substance and relatively draw quantitative values; The PCR primer of said miRNA-483-5p and probe are like following table: shown in

.

2. the quantitative detecting method of serum miRNA-483-5p as claimed in claim 1 is characterized in that in step 1), and said serum to be detected is 400 μ l.

3. the quantitative detecting method of serum miRNA-483-5p as claimed in claim 1 is characterized in that in step 1), the consumption of said lysate and the same volume of serum to be detected.

4. the quantitative detecting method of serum miRNA-483-5p as claimed in claim 1 is characterized in that in step 2) in, the said quantitative PCR detection of carrying out adopts ABI7300 quantitative PCR appearance.