CN105950708A - Fluorogenic quantitative PCR detection primer and method for miR-100 content - Google Patents

Fluorogenic quantitative PCR detection primer and method for miR-100 content Download PDF

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CN105950708A
CN105950708A CN201610195171.3A CN201610195171A CN105950708A CN 105950708 A CN105950708 A CN 105950708A CN 201610195171 A CN201610195171 A CN 201610195171A CN 105950708 A CN105950708 A CN 105950708A
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mir
primer
pcr
content
reverse transcription
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章晓波
龚燚
杨耿
王雨濛
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Shanghai and Jun Huxing Biotechnology Co., Ltd.
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention discloses a fluorogenic quantitative PCR detection primer and method for a miR-100 content. The miR-100 content of a sample is detected through fluorogenic quantitative PCR. The method can fast and accurately detect the miR-100 content of a detected sample. Since the miR-100 content in pathological tissues and serum is obviously increased along with patients attacked by gastric carcinoma, quick effective detection of miR-100 is of great significance to monitoring of the patients.

Description

The primer of a kind of fluorescence quantitative PCR detection miR-100 content and detection method
(1) technical field
The present invention relates to the detection of miR-100, particularly to fluorescence quantitative PCR detection primer.
(2) background technology
Gastric cancer is the malignant tumor that a kind of digestive system is common, and sickness rate is high, and in China, fatality rate occupies first of various malignant tumor, and discovery and the application of stomach cancer marker have great meaning for the early diagnostic rate improving gastric cancer.MicroRNAs (miRNAs) is the class non-coding tiny RNA being found recently, research shows, the generation of most cancers is all relevant with the Abnormal regulation of miRNA, the content of miRNA there occurs significantly change, wherein, this laboratory finds, the morbidity of patients with gastric cancer is all along with miR-100 notable rise of content in pathological tissues and serum, therefore, in the therapeutic process of patients with gastric cancer, monitoring to miR-100 content becomes extremely important, is badly in need of setting up one miR-100 detection method fast and accurately.
(3) summary of the invention
It is an object of the present invention to provide primer and the detection method of a kind of fluorescence quantitative PCR detection miR-100 content.
The technical solution used in the present invention is:
The present invention provides the primer of a kind of fluorescence quantitative PCR detection miR-100 content, and described primer includes miR-100 reverse transcription primer and miR-100 fluorescence quantification PCR primer F and primer R；
The nucleotides sequence of described miR-100 is classified as 5 '-AACCCGUAGAUCCGAACUUGUG-3 ',
The nucleotides sequence of described miR-100 reverse transcription primer is classified as:
5 '-GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACCACAAGT-3 ',
The nucleotides sequence of described miR-100 fluorescence quantification PCR primer F is classified as:
5 '-CGCCGAACCCGTAGATCCG-3 ',
The nucleotides sequence of described miR-100 fluorescence quantification PCR primer R is classified as:
5’-TGCAGGGTCCGAGGTCACTG-3’。
The present invention also provides for a kind of method of primer detection miR-100 content utilizing described fluorescence quantitative PCR detection miR-100 content, and described method is:
(1) extract the miRNA in testing sample, utilize miRNA Reverse Transcription (AB company) and miR-100 reverse transcription primer that by PCR reaction reverse transcription, the miRNA of extraction is become cDNA；The response procedures of described reverse transcriptional PCR is: 16 DEG C are reacted 30 minutes, and 42 DEG C are reacted 30 minutes, and last 85 DEG C are reacted 5 minutes；
(2) with reverse transcription product cDNA as template, quantitative fluorescent PCR reagent (AB company) and miR-100 fluorescence quantification PCR primer is utilized to carry out PCR reaction；The response procedures of described quantitative fluorescent PCR is: 95 DEG C reaction 10 minutes after, carry out 50 circulation 95 DEG C 5 seconds, 60 DEG C 1 minute.
(3) method using absolute quantitation, testing sample in step (1) is changed into the mixed liquor containing miR-100 standard substance and carries out reverse transcription and quantitative fluorescent PCR reaction, according to the CT value of taking off that the concentration of miR-100 standard substance in the mixed liquor containing miR-100 standard substance is corresponding, obtain miR-100 content and react the linear relationship between CT value with quantitative fluorescent PCR, be depicted as the standard curve between concentration and CT value；According to testing sample CT value, i.e. can detect that the content of miR-100.
Testing sample of the present invention includes: cell, tissue or serum.
Total RNA extraction reagent is used to extract the miRNA in testing sample, if tissue, add Trizol after adding a small amount of liquid nitrogen grinding to crack, if attached cell or serum, the most direct Trizol carries out digestion cracking, after cracking, 12000rpm is centrifuged five minutes, abandon precipitation, the concussion mixing of rear addition chloroform stands 15 minutes, 4 DEG C of centrifugal 15min, fetch water to new centrifuge tube, add isopropanol mixing, place 5-10 minute, centrifugal, abandon supernatant, RNA is i.e. sunken at the bottom of pipe, add the water dissolution RNA sample of 50 μ L, place for 55-60 DEG C and carry out next step reaction after 5 minutes.
nullThe beneficial effects are mainly as follows: this method is compared with existing detection method，Owing to being the specific detection experiment of miR-100 content，The specific reverse transcription of so miR-100 is the most key with the primer of quantitative PCR，The primer of miRNA in the past all relies on the import of American I nvitrogen company，The most expensive (single pair of primer price surpasses 2000 RMB)，And due to from external import，Delivery date length (time is no less than one month)，Therefore the detection of miRNA content inconvenience very is carried out，The primer of the specific reverse transcription and quantitative PCR that the present invention is directed to the most key miR-100 of this experiment is started with，Pass through the experiment to miRNA loop-stem structure and analysis，The specific reverse transcription of miR-100 and the primer sequence of quantitative PCR are successfully obtained，Subsequent experimental demonstrates the reliability of this primer，And delivery date short (domestic synthesis 1 to 2 days)，Low price (tens RMB)，This invention is that the research of miRNA is provided convenience.
The present invention provides the primer of a kind of fluorescence quantitative PCR detection miR-100 content, utilize miR-100 content in fluorescence quantitative PCR detection sample, the inventive method can detect the content of miR-100 in sample fast and accurately, owing to the morbidity of patients with gastric cancer is all along with miR-100 notable rise of content in pathological tissues and serum, therefore, miR-100 detection fast and effectively has great importance for the monitoring of patients with gastric cancer.
(4) accompanying drawing explanation
Fig. 1 be screening verification miR-100 primer whether can quantitative PCR solubility curve.
Fig. 2 is standard curve.
Fig. 3 utilizes primer of the present invention detection stomach organization and the result figure of miR-100 content in normal structure.
Fig. 4 utilizes primer of the present invention detection Serum Obtained From Advance Gastric Cancer and the result figure of miR-100 content in normal serum.
Fig. 5 is miRNA stem ring.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited to that:
Embodiment 1:
According to the laboratory order-checking to miRNA stem ring (Fig. 5), transcribe mechanism in conjunction with miRNA, have devised reverse transcription and the quantification PCR primer of miRNA.
Reverse transcription primer:
GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACXXXXXXX
XXXXXXX represents the sequence obtained by last for miR-100 mature sequence 7 base reverse complementals.
Quantitative PCR forward primer: CGCCGXXXXXXXXXXXXX
XXXXXXXXXXXXX represents front 13 base sequences of miR-100 mature sequence.
Quantitative PCR reverse primer: TGCAGGGTCCGAGGTCACTG
Utilize this design of primers rule, can be designed that the primer of any miRNA.
The checking of miR-100 primer
(1) miR-100 standard substance DEPC water is configured to 10-3、10-2、10-1、100、101、102、103μM mi-100 standard solution, utilize miRNA Reverse Transcription (AB company) and miR-100 reverse transcription primer by the miRNA of extraction by PCR reaction reverse transcription become cDNA；The response procedures of described reverse transcriptional PCR is: 16 DEG C are reacted 30 minutes, and 42 DEG C are reacted 30 minutes, and last 85 DEG C are reacted 5 minutes；
(2) with reverse transcription product cDNA as template, quantitative fluorescent PCR reagent (AB company) and miR-100 fluorescence quantification PCR primer is utilized to carry out PCR reaction；The response procedures of described quantitative fluorescent PCR is: 95 DEG C reaction 10 minutes after, carry out 50 circulation 95 DEG C 5 seconds, 60 DEG C 1 minute.
As shown in Figure 1, PCR solubility curve is the most single for result, illustrates that primer specificity is good, can effectively detect miR-100 content.
Embodiment 2 standard curve
(1) miR-100 standard substance DEPC water is configured to 10-3、10-2、10-1、100、101、102、103μM mi-100 standard solution, utilize miRNA Reverse Transcription (AB company) and miR-100 reverse transcription primer by the miRNA of extraction by PCR reaction reverse transcription become cDNA；The response procedures of described reverse transcriptional PCR is: 16 DEG C are reacted 30 minutes, and 42 DEG C are reacted 30 minutes, and last 85 DEG C are reacted 5 minutes；
(2) with reverse transcription product cDNA as template, quantitative fluorescent PCR reagent (AB company) and miR-100 fluorescence quantification PCR primer is utilized to carry out PCR reaction；The response procedures of described quantitative fluorescent PCR is: 95 DEG C reaction 10 minutes after, carry out 50 circulation 95 DEG C 5 seconds, 60 DEG C 1 minute.According to the take off CT value corresponding containing the concentration of miR-100 standard substance in miR-100 standard solution, obtaining miR-100 content and react the linear relationship between CT value with quantitative fluorescent PCR, be depicted as the standard curve between concentration and CT value, result is as shown in Figure 2.
CT=3.259 × lgx+33.777, x are miR-100 content in miR-100 standard solution.
The nucleotides sequence of described miR-100 is classified as 5 '-AACCCGUAGAUCCGAACUUGUG-3 ',
The nucleotides sequence of described miR-100 reverse transcription primer is classified as:
5 '-GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACCACAAGT-3 ',
The nucleotides sequence of described miR-100 fluorescence quantification PCR primer F is classified as:
5 '-CGCCGAACCCGTAGATCCG-3 ',
The nucleotides sequence of described miR-100 fluorescence quantification PCR primer R is classified as:
5’-TGCAGGGTCCGAGGTCACTG-3’。
The detection method of embodiment 3 testing sample:
Testing sample: patients with gastric cancer gastric tissue, the acquisition of Serum Obtained From Advance Gastric Cancer complies fully with the requirement of medical science or ethics, obtains the most within the hospital, is the agreement having obtained patient.
(1) miRNA in testing sample is extracted, miRNA Reverse Transcription (AB company) and miR-100 reverse transcription primer is utilized by PCR reaction reverse transcription, the miRNA of extraction to be become cDNA, with the miRNA Reverse Transcription (AB company) that comprises primer for compareing；The response procedures of described reverse transcriptional PCR is: 16 DEG C are reacted 30 minutes, and 42 DEG C are reacted 30 minutes, and last 85 DEG C are reacted 5 minutes；
(2) with reverse transcription product cDNA as template, quantitative fluorescent PCR reagent (AB company) and miR-100 fluorescence quantification PCR primer is utilized to carry out PCR reaction, with the quantitative fluorescent PCR reagent (AB company) that comprises primer for comparison；The response procedures of described quantitative fluorescent PCR is: 95 DEG C reaction 10 minutes after, carry out 50 circulation 95 DEG C 5 seconds, 60 DEG C 1 minute.
(3) standard curve made according to embodiment 2；According to testing sample CT value, i.e. can detect that the content of miR-100, result is shown in Fig. 3 and Fig. 4.
Total RNA extraction reagent is used to extract the miRNA in testing sample, if tissue, add Trizol after adding a small amount of liquid nitrogen grinding to crack, if attached cell or serum, the most direct Trizol carries out digestion cracking, after cracking, 12000rpm is centrifuged five minutes, abandon precipitation, the concussion mixing of rear addition chloroform stands 15 minutes, 4 DEG C of centrifugal 15min, fetch water to new centrifuge tube, add isopropanol mixing, place 5-10 minute, centrifugal, abandon supernatant, RNA is i.e. sunken at the bottom of pipe, add the water dissolution RNA sample of 50 μ L, place for 55-60 DEG C and carry out next step reaction after 5 minutes.
Fig. 3 is totally 57 example of miR-100 content ＞ 100fM in patients with gastric cancer gastric tissue, totally 8 example of miR-100 content ＜ 100fM, in non-patients with gastric cancer tissue, miR-100 content ＞ 100fM's has 11 examples, and miR-100 content ＜ 100fM has 55 examples.Specificity is 83.8% (57/68), and sensitivity is 86.4% (57/66).With 0.1fM as marginal value, in serum miR-100 content more than in 0.1fM or gastric tissue more than 100fM, can effectively differentiate patients with gastric cancer and normal person.
Fig. 4 is totally 24 example of miR-100 content ＞ 0.1fM in Serum Obtained From Advance Gastric Cancer, and totally 5 example of miR-100 content ＜ 0.1fM, in non-Serum Obtained From Advance Gastric Cancer, miR-100 content ＞ 0.1fM does not has, all less than 0.1fM.MiR-100 is 100% (24/24) as the specificity of diagnosis index, and sensitivity is 82.8% (24/29).These conclusions below are the results worked it out with the primer of the present invention, compared with the existing methods, it it is only the difference of primer, and the checking that this primer is by solubility curve, it is possible to the special expression effectively detecting miR-100, the reliability of this primer is described, as for these conclusions, all being because this miRNA of miR-100 in cancer patient with normal person is differential expression, and whether with the import primer bought or the primer of the present invention, the conclusion obtained should be all consistent.
With 0.1fM as marginal value, in serum miR-100 content more than in 0.1fM or gastric tissue more than 100fM, can effectively differentiate patients with gastric cancer and normal person.

Claims (3)

1. the primer of a fluorescence quantitative PCR detection miR-100 content, it is characterised in that described primer Including miR-100 reverse transcription primer and miR-100 fluorescence quantification PCR primer F and primer R；
The nucleotides sequence of described miR-100 is classified as 5 '-AACCCGUAGAUCCGAACUUGUG-3 ',
The nucleotides sequence of described miR-100 reverse transcription primer is classified as:
5’-GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACCA
CAAGT-3 ',
The nucleotides sequence of described miR-100 fluorescence quantification PCR primer F is classified as:
5 '-CGCCGAACCCGTAGATCCG-3 ',
The nucleotides sequence of described miR-100 fluorescence quantification PCR primer R is classified as:
5’-TGCAGGGTCCGAGGTCACTG-3’。
2. the primer utilizing fluorescence quantitative PCR detection miR-100 content described in claim 1 is examined The method surveying miR-100 content, it is characterised in that described method is:
(1) extract the miRNA in testing sample, utilize miRNA Reverse Transcription and miR-100 The miRNA of extraction is become cDNA by PCR reaction reverse transcription by reverse transcription primer；Described reverse transcriptional PCR Response procedures be: 16 DEG C react 30 minutes, 42 DEG C react 30 minutes, last 85 DEG C react 5 minutes；
(2) with reverse transcription product cDNA as template, quantitative fluorescent PCR reagent and miR-100 are utilized Fluorescence quantification PCR primer carries out PCR reaction；The response procedures of described quantitative fluorescent PCR is: 95 DEG C After reacting 10 minutes, carry out 50 circulation 95 DEG C 5 seconds, 60 DEG C 1 minute.
(3) method using absolute quantitation, changes into testing sample in step (1) preparing with DEPC water MiR-100 standard solution carry out reverse transcription and quantitative fluorescent PCR reaction, according to containing miR-100 mark CT value that what in the mixed liquor of quasi-product, the concentration of miR-100 standard substance was corresponding take off, obtains miR-100 and contains Amount reacts the linear relationship between CT value with quantitative fluorescent PCR, is depicted as between concentration and CT value Standard curve；According to testing sample CT value, i.e. can detect that the content of miR-100.
3. method as claimed in claim 2, it is characterised in that described testing sample includes: cell, group Knit or serum.
CN201610195171.3A 2016-03-31 2016-03-31 Fluorogenic quantitative PCR detection primer and method for miR-100 content Pending CN105950708A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105176983A (en) * 2015-09-23 2015-12-23 河南师范大学 Kit for detecting esophageal squamous carcinoma associated serum miRNAs genes
CN105219867A (en) * 2015-11-02 2016-01-06 杨廷稳 For miRNA biomarker and the detection kit of diagnosing gastric cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105176983A (en) * 2015-09-23 2015-12-23 河南师范大学 Kit for detecting esophageal squamous carcinoma associated serum miRNAs genes
CN105219867A (en) * 2015-11-02 2016-01-06 杨廷稳 For miRNA biomarker and the detection kit of diagnosing gastric cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHU ZHOU等: "Prognostic value of microRNA-100 in esophageal squamous cell carcinoma", 《JOURNAL OF SURGICAL RESEARCH》 *
宋伟等: "基于茎环引物定量检测miRNA的研究进展", 《食品工业科技》 *

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Address after: 201201 206, room 1, 6 Jinfeng Road, Tang Town, Pudong New Area, Shanghai.

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Application publication date: 20160921