CN108660213B - Application of reagent for detecting three non-coding RNAs and kit - Google Patents
Application of reagent for detecting three non-coding RNAs and kit Download PDFInfo
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Abstract
The invention discloses an application of a reagent for detecting three non-coding RNAs and a kit. In particular to a preparation for assisting the diagnosis of tumors by a real-time fluorescence quantitative analysis method for detecting circular RNA circMAN1A2, circular RNA circRNF13 and long-chain non-coding RNA LOC 284454. Research proves that the expression of circMAN1A2, circRNF13 and LOC284454 is up-regulated in the serum of patients with nasopharyngeal carcinoma, oral cancer and thyroid cancer. Therefore, the expression conditions of circMAN1A2, circRNF13 and LOC284454 are jointly used for auxiliary diagnosis of tumors, the diagnosis accuracy is further improved, and the method has profound clinical significance and important popularization and application prospects.
Description
Technical Field
The invention belongs to the technical field of tumor molecular biology, and particularly relates to a method for detecting circular RNA
The application of reagents of circMAN1A2, circular RNA circRNF13 and long non-coding RNA LOC284454 in preparing tumor auxiliary diagnostic preparations and corresponding kits.
Background
Malignant tumors seriously harm human health, and the incidence rate of Chinese malignant tumors is on the rise in recent years. According to statistics of Chinese cancer statistical center, the number of new malignant tumors is about 380.4 ten thousand, wherein 211.4 ten thousand of male patients and 169.0 ten thousand of female patients can be diagnosed as cancer about more than 1 ten thousand times per day, and about 7 times per minute. The incidence rate of the malignant tumor is 278.07/10 ten thousand, wherein the incidence rate of the malignant tumor in men is 301.67/10 ten thousand, and the incidence rate of the malignant tumor in women is 253.29/10 ten thousand. The average rate of the incidence rate of the malignant tumor is 190.63/10 ten thousand (the population standard rate is based on the standard population structure of China in 2000), the world standard rate of the incidence rate of the malignant tumor is 186.53/10 ten thousand (the population standard rate is based on the Segi's world standard population structure), and the cumulative incidence rate of 0-74 years is about 21.58%. The mortality rate of malignant tumor is about 167.89/10 ten thousand, wherein the medium rate is about 106.98/10 ten thousand, the world standard rate is about 106.09/10 ten thousand, and the cumulative mortality rate of 0-74 years is about 12.00%.
According to the statistics of the American Cancer Society (ACS), the number of new diseases of American malignant tumors is estimated to be about 173.5 ten thousands, and 4700 new diseases are all found every day. The incidence of malignancy in the united states steadily decreases, with mortality rates for malignancy decreasing at a rate of 1.5% per year. The significant decline in the incidence of malignant tumors in the united states may be attributed to the control of smoking, the promotion of multi-level cancer screening, and the widespread use of novel cancer therapies.
It is thus clear that the reduction of the causative factors of malignant tumors, the effective early diagnosis, and the search for novel cancer therapies play a significant role in the control of cancer. Therefore, in the medical research of China, the research is dedicated to searching for novel tumor markers in order to provide a new way for early diagnosis of malignant tumors and for early, convenient and accurate diagnosis of diseases so as to be beneficial to treatment.
Circular RNA (circRNA) is a new class of RNA molecules with a configuration that is different from conventional linear RNA (containing 5 'and 3' ends), and circular RNA molecules are in a closed circular structure (i.e. 5 'and 3' ends end to end), and circular RNA is more stable than linear RNA because there are no free 5 'and 3' ends. At first, circRNA is considered as a byproduct of missplicing and is not widely concerned, in recent years, the wide application of RNA-Sequencing based on a new generation Sequencing technology promotes the rapid development of many fields of RNA, and a large amount of new circRNA is found, so that the circRNA is pushed to the stage center of malignant tumor genomics research.
As an emerging RNA, circRNA, its mechanism of action and function is attracting increasing research. As more and more circrnas were identified, some were found to be expressed at levels much higher than their associated linear genes. The circRNAs form a circular structure by covalent bonds, and most of the circRNAs are reverse splicing products, are widely expressed in organisms, sometimes have the expression level 10 times that of linear RNAs, and are mostly localized in cytoplasm. The CircRNA can be used as a molecular sponge of miRNA to adsorb miRNA, bind protein, or be used as a regulatory factor of gene transcription and protein translation, and the like to play important biological functions, is likely to become a new biomarker and a clinical treatment target of malignant tumor, and provides a new idea for diagnosis and prognosis of tumor.
Long non-coding RNAs (lncRNAs) are RNAs molecules which are over 200nt long, lack a specific complete open reading frame and have no or few protein coding functions. Recent studies have shown that lncRNAs are involved in the regulation of important vital activities such as growth, development, senescence and death of organisms by regulating gene expression at epigenetic, transcriptional and post-transcriptional levels. More and more researches show that the abnormal expression or the function loss of the lncRNAs are closely related to the occurrence and the development of tumors. Some lncRNAs have important significance in the diagnosis and treatment of tumors and can be used as a new molecular marker for judging tumor prognosis.
Therefore, more novel non-coding RNAs need to be screened and verified as biomarkers for cancer diagnosis and prognosis and application thereof, and can be well protected in the patent field as soon as possible, and the international competitiveness of China in the technical field can be remarkably improved.
Disclosure of Invention
According to the invention, high-expression circRNA and IncRNA are screened from RNA seq data, the expression conditions of the circRNA and the IncRNA are verified in various types of cancers respectively, and the emerging circRNA and IncRNA can be used as serum molecular markers for diagnosing malignant tumors.
Therefore, the first purpose of the invention is to provide an application of a reagent for detecting three non-coding RNAs, wherein the reagent is used for preparing a tumor auxiliary diagnostic preparation, the three non-coding RNAs are circular RNA circMAN1A2, the sequence of the circular RNA circRNF13 is shown as SEQ No.2, and the sequence of the long-chain non-coding RNA LOC284454 is shown as SEQ No. 3. Provides a new accurate, reliable, simple and convenient detection way for the auxiliary diagnosis of the tumor.
Compared with tissue detection, the serum is used as a detection sample, and has the advantages of convenient sample acquisition mode and operation, low cost, high accuracy and ideal diagnosis for tumors. Therefore, the applicant uses serum as a sample in the research process, and searches and verifies the molecules in the serum relevant to tumor diagnosis and the relationship between the molecules and the tumor through a large number of tests. The applicant firstly discovers that the relationship of circular RNA circMAN1A2, circular RNA circRNF13 and long-chain non-coding RNA LOC284454 existing in serum and various tumors is positively correlated, and the relationship can be conveniently used for assisting or primarily diagnosing the possibility of having the tumors by detecting the content of the circular RNA circMAN1A2, the circular RNA circRNF13 and the long-chain non-coding RNA LOC284454 in the serum.
According to the invention, an experiment method of SYBR-qPCR or Taqman probe q-PCR is adopted to successively verify the expression conditions of circMAN1A2, circRNF13 and LOC284454 in nasopharyngeal carcinoma, oral cancer and thyroid cancer respectively.
The invention is used for verifying that the samples collected by the expression conditions of circMAN1A2, circRNF13 and LOC284454 are sufficient and have statistical significance, and the samples have reasonable sources, strict screening standards, strict test process and real and reliable results.
The test result shows that the expressions of circMAN1A2, circRNF13 and LOC284454 in the serums of patients with nasopharyngeal carcinoma, oral cancer and thyroid cancer are all obviously up-regulated, the consistency of the expression trends in various tumors is shown, and the results indicate that the circMAN1A2, circRNF13 and LOC284454 molecules can be respectively and independently used as molecular markers for tumor diagnosis, but the statistical analysis shows that the three non-coding RNAs are combined to judge together, and the accuracy is higher.
In the application, the reagent for detecting the three non-coding RNAs comprises a real-time fluorescent quantitative detection reagent.
In order to ensure the specificity of the primers and the accuracy of qPCR, the applicant designs the primers which can accurately amplify the expression of the circular RNA MAN1A2 for real-time fluorescent quantitative detection by strictly following the RNA primer design principle:
an upstream primer: 5'-AGATGGGCAAAGATGGATTGA-3'
A downstream primer: 5'-GCCTTCTCATGATCAGCTCG-3', respectively;
and a primer for real-time fluorescent quantitative detection of the expression of the circular RNA RNF 13:
an upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3' are provided.
And a primer for real-time fluorescent quantitative detection of long-chain non-coding RNA LOC284454 expression:
an upstream primer: 5'-ATTACAGGTGGCTCAGGTGT-3'
A downstream primer: 5'-CTTCAGTGTGCCTCCTCAGT-3'
The second purpose of the invention is to provide a kit for tumor auxiliary diagnosis; provides a new accurate and reliable detection product for the tumor auxiliary diagnosis.
The kit contains a reagent capable of detecting circular RNA circMAN1A2, circular RNA circRNF13 and long-chain non-coding RNA LOC284454 in serum; the sequence of the circular RNA circMAN1A2 is shown as SEQ No.1, the sequence of the circular RNA circRNF13 is shown as SEQ No.2, and the sequence of the long non-coding RNA LOC284454 is shown as SEQ No. 3.
Further, the primer capable of amplifying the circular RNA circMAN1A2 is preferably the following primer:
an upstream primer: 5'-AGATGGGCAAAGATGGATTGA-3'
A downstream primer: 5'-GCCTTCTCATGATCAGCTCG-3' are provided.
And the primer capable of amplifying the circular RNA circRNF13 is preferably the following primer:
an upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3' are provided.
A primer capable of amplifying long-chain non-coding RNA LOC 284454:
an upstream primer: 5'-ATTACAGGTGGCTCAGGTGT-3'
A downstream primer: 5'-CTTCAGTGTGCCTCCTCAGT-3'
However, the primers of the present invention capable of amplifying circular RNAs circMAN1A2 and circRNF13, and long non-coding RNA LOC284454 are not limited to the primers provided above.
Because human serum has no internal reference gene, the PGL3 plasmid is preferably added as a reference to reduce errors in the RNA extraction process, such as differences in RNA adsorption efficiency and elution efficiency, and provide more reliable experimental data for research. Because of the quantitative addition of GL3 plasmid, the corresponding copy number of the target gene can be calculated from the ct value obtained by q-PCR reaction, thereby achieving absolute quantification. The addition of the external reference gene reduces the system error in the experiment, thereby providing data information with higher reliability for research.
Therefore, the temperature of the molten metal is controlled,
further, the kit of the present invention further comprises: internal control pGL3 primer:
an upstream primer: 5'-TCCATCTTGCTCCAACACCC-3'
A downstream primer: 5'-TCGTCTTTCCGTGCTCCAAA-3' are provided.
However, the reference primer of the present invention is not limited to the reference pGL3 primer provided above.
The Taqman probe has high specificity, high sensitivity and good specificity. The SYBR method needs to carry out PCR reaction in different EP tubes, while the Taqman probe method can realize the simultaneous detection of a plurality of genes in one EP tube, thereby reducing the experimental error. The Taqman probe-qPCR experimental method is rigorous, has high reliability and repeatability, can simultaneously detect various samples, has good experimental repeatability, obtains more reliable data, and provides a solid basis for the evaluation of clinical application value.
Therefore, the temperature of the molten metal is controlled,
further, the kit of the present invention preferably further comprises: the Taqman probe matched with the primer for real-time fluorescent quantitative detection of circular RNA circMAN1A2 expression comprises the following components: 5 '-ROX-CAAAGATGGATTGAAGACAACCTTGATTTCAGTGTG-BHQ 2-3'.
And a Taqman probe matched with the primer for real-time fluorescent quantitative detection of circular RNA circRNF13 expression: 5 '-Cy 5-AGGATAGACATAGAGCTAGAAGAAACAGACTTCGT-BHQ 2-3'.
The Taqman probe matched with the primer for real-time fluorescent quantitative detection of long-chain non-coding RNA LOC284454 expression comprises the following components:
5’-FAM–CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3’
further, the kit of the present invention preferably further comprises: a Taqman probe matched with an internal reference pGL3 primer: 5 '-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ 1-3'.
However, the Taqman probe used in the present invention is not limited to the above-mentioned kit probe.
Further, the kit of the present invention further comprises: a serum total RNA extraction reagent, an RNA reverse transcription PCR reaction reagent and a real-time fluorescent quantitative detection reagent.
The invention extracts RNA from the serum of various tumor patients and the serum of normal human, carries out reverse transcription, and detects the expression of circMAN1A2, circRNF13 and LOC284454 by a real-time fluorescence quantitative method, and the result shows that the expression of the circMAN1A2, the circRNF13 and the LOC284454 is up-regulated in the serum of various tumor patients. Circular RNA molecules circMAN1A2 and circRNF13 exist in serum for the first time, and long-chain non-coding RNA LOC284454 and various tumors exist a uniform forward correlation relationship, so that the circMAN1A2, the circRNF13 and the LOC284454 can be used as molecular markers for tumor auxiliary diagnosis, and particularly the combination of the circular RNA molecules circMAN1A2 and the circRNF13 has higher accuracy. The invention provides a powerful molecular biology tool for the auxiliary diagnosis of tumors, and has profound clinical significance and important popularization and application prospects.
Drawings
FIG. 1 is a circMAN1A2 and circRNF13 map;
in the figure, a is circMAN1A2, and b is circRNF 13.
FIG. 2 shows the reference plasmid pGL 3Q-PCR standard curve.
FIG. 3 shows SYBR-qPCR detection of LOC284454, circRNF13, circMAN1A2 expression in serum of nasopharyngeal carcinoma patients;
in the figure, a, b and c are respectively SYBR-qPCR detection of the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of a nasopharyngeal carcinoma patient, wherein compared with a normal control group, the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of the nasopharyngeal carcinoma patient are up-regulated, and P is less than 0.001; n represents a normal control group, T represents a nasopharyngeal carcinoma group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 4 is a Taqman-qPCR assay for the expression of LOC284454, circRNF13 and circMAN1A2 in the serum of nasopharyngeal carcinoma patients;
in the figure, a, b and c are respectively Taqman-qPCR detection of the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of a nasopharyngeal carcinoma patient, wherein the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of the nasopharyngeal carcinoma patient are up-regulated compared with a normal control group, and P is less than 0.001; n represents a normal control group, T represents a nasopharyngeal carcinoma group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 5 is a nasopharyngeal carcinoma group ROC curve;
in the figure, a, b, c and d are ROC curves of LOC284454, circRNF13, circMAN1A2 and LOC284454 in combination with circMAN1A2 and circRNF13 respectively.
FIG. 6 is a Taqman-qPCR assay for the expression of LOC284454, circRNF13 and circMAN1A2 in the serum of patients with oral cancer;
in the figure, a, b and c are respectively Taqman-qPCR detection of the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of an oral cancer patient, wherein the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of the oral cancer patient are up-regulated compared with a normal control group, and P is less than 0.001; n represents a normal control group, T represents an oral cancer group, and N represents the number of samples. P <0.05, P <0.01, P < 0.001.
FIG. 7 is a ROC curve for the oral cancer group;
in the figure, a, b, c and d are ROC curves of LOC284454, circRNF13, circMAN1A2, LOC284454 combined with circMAN1A2 and circRNF13 respectively.
FIG. 8 is Taqman-qPCR assay for the expression of LOC284454, circRNF13 and circMAN1A2 in the serum of thyroid cancer patients;
in the figure, a, b and c are respectively Taqman-qPCR detection of the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of a thyroid cancer patient, wherein the expression levels of LOC284454, circRNF13 and circMAN1A2 in the serum of the thyroid cancer patient are up-regulated compared with a normal control group, and P is less than 0.001; n represents a normal control group, T represents a thyroid cancer group, and N represents the number of samples; p <0.05, P <0.01, P < 0.001.
FIG. 9 is a thyroid cancer panel ROC curve;
in the figure, a, b, c and d are ROC curves of LOC284454, circRNF13, circMAN1A2, LOC284454 combined with circMAN1A2 and circRNF13 respectively.
Detailed Description
The following detailed description is intended to further illustrate the invention and is not to be construed as limiting the invention.
The normal control group and the tumor group serum samples adopted in the invention are respectively from the Hunan Yabi hospital examination center and the Hunan province tumor hospital examination center of the university in the south China. 121 cases are collected in the normal control group, and the tumor diseases, infectious diseases, serious immune diseases and other serious diseases are eliminated; tumor group serum samples included: 100 serum samples of nasopharyngeal carcinoma patients, 55 serum samples of oral cancer patients, 57 serum samples of thyroid cancer patients, and blood collection time interval: 3 months in 2017 to 1 month in 2018. The collected blood samples gradually complete clinical data, including patient name, sex, age, hospitalization number, pathological type, pathological stage, identity card number, treatment condition, etc. All serum samples were informed by the patient or the person who collected the serum samples, and a blood sample bank with complete clinical data was gradually established.
1. Realtime PCR primer
The primers and probes used in the invention are designed through a special Primer design website Primer 3.0. The primer synthesis work entrusted the synthesis of the department of Engineers. To ensure primer specificity and qPCR accuracy, we strictly followed the following primer design principles:
the design principle of the probe is as follows:
the primer and the Taqman probe used by the invention have the following sequences:
(1) pGL3 primer
An upstream primer: 5'-TCCATCTTGCTCCAACACCC-3', respectively;
a downstream primer: 5'-TCGTCTTTCCGTGCTCCAAA-3', respectively;
taqman probe: 5 '-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ 1-3';
(2) LOC284454 primer
An upstream primer: 5'-ATTACAGGTGGCTCAGGTGT-3'
A downstream primer: 5'-CTTCAGTGTGCCTCCTCAGT-3'
Taqman probe
5’-FAM–CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3’
(3) circMAN1A2 primer
An upstream primer: 5'-AGATGGGCAAAGATGGATTGA-3', respectively;
a downstream primer: 5'-GCCTTCTCATGATCAGCTCG-3', respectively;
taqman probe: 5' -ROX-
CAAAGATGGATTGAAGACAACCTTGATTTCAGTGTG-BHQ2-3’；
(4) circRNF13 primer
An upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3'
Taqman probe: 5 '-Cy 5-AGGATAGACATAGAGCTAGAAGAAACAGACTTCGT-BHQ 2-3';
CircMAN1a2 and CircRNF13, see fig. 1.
2 method
2.1 cancer patient peripheral blood sample Collection
The sample collection procedure was as follows:
(1) collecting peripheral blood samples by using an EDTA (ethylene diamine tetraacetic acid) anticoagulation blood collection tube, collecting 1-2 ml of fasting venous blood, and turning the blood collection tube upside down to mix the blood samples gently and uniformly so as to avoid hemolysis;
(2) centrifuging the whole blood sample at 4 ℃ for 10min at 1600g, extracting 500-;
(3) immediately placing the plasma specimen in a refrigerator at-80 ℃ for storage after extraction, and finishing the whole process after blood separation within 4 hours;
(4) filling in 'sample information record table', collecting the information of patient and normal person's name, sex and age, and the clinical data of patient's hospital number, pathological type, pathological stage, identity card number and treatment.
2.2 serum Total RNA extraction
The method adopts a miRNeasy Serum/plasmaikit kit, and comprises the following specific steps:
(1) 200ul plasma was placed in 2ml EP tubes;
(2) add 5 times volume of lysis buffer (i.e. 1000ul) to EP tube, vortex or mix well with pipette;
(3) the dissolved product in the EP tube is placed for 5min at room temperature (15-25 ℃);
(4) adding radix Ginseng PGL3, and mixing (1.6 x 10)8copies/ul)；
(5) Add equal sample volume of chloroform (i.e. 200ul), vortex or shake vigorously for 15 s;
(6) standing at room temperature (15-25 deg.C) for 2-3 min;
(7) centrifuging 12000g at 4 ℃ for 15min, adjusting the temperature to room temperature after using the centrifuge, and obtaining an upper colorless water phase of RNA; the middle white layer is DNA; the lower red organic phase is protein;
(8) sucking the upper water phase into a new 2ml EP tube (600ul), adding 1.5 times of 100% alcohol (900 ul), mixing well with a pipette, adding alcohol to form precipitate without influence;
(9) placing the column into a 2ml tube, sucking 700ul of the column, closing the cover, centrifuging at room temperature (15-25 deg.C) for 15s at more than or equal to 8000g (10000rpm), and discarding the waste liquid in the collection tube;
(10) repeating (9), passing the residual sample through the column, and discarding the waste liquid in the collection tube;
(11) adding 700ul Buffer RWT to the column, closing the cover, centrifuging to more than or equal to 8000g (10000rpm) for 15s, and discarding the waste liquid in the collection tube;
(12) adding 500ul Buffer RPE to the column, closing the cover, centrifuging for 15s at a speed of more than or equal to 8000g (10000rpm), and discarding the waste liquid in the collection tube;
(13) adding 500ul 80% ethanol into the column, slightly closing the cover, centrifuging to more than or equal to 8000g (10000rpm), washing the column for 2min, and discarding the collecting tube (carefully moving the column to avoid getting on the waste liquid of the collecting tube);
(14) placing the column in a new 2ml Tube, opening the lid, centrifuging at full speed for 5min, drying, discarding the collection Tube (to avoid damaging the lid, the Tube is placed at an interval such that the lid faces upwards and the rotor turns in the opposite direction);
(15) place the column in a fresh 1.5ml Tube, add 14ul of enzyme-free water to the center of the column membrane, gently close the column, centrifuge at full speed for 1min, and elute the RNA to about 12ul in a 1.5ml Tube.
We added the PGL3 plasmid as a reference, providing more reliable experimental data for the present invention.
The reference plasmid was added at a concentration of about 2X 10 pGL3 per ml of serum8Copies were calculated to be approximately 1ng plasmid per ml serum;
a10. mu.l reaction for Real time PCR was as follows:
the pGL3 plasmid standard curve was plotted as follows: 1ng plasmid is taken and added with enzyme-free water to be constant volume to 500 mul, and then 2 mul plasmid is taken and added with enzyme-free water to be constant volume to 1ml to be used as a 1 st tube; then gradually diluting in half, i.e.: 500ul of plasmid solution is taken from the 2 nd tube and put into the 2 nd tube, 500ul of non-enzyme water is added and mixed evenly; remove 500ul plasmid solution from the 2 nd tube and put into the third tube, add 500ul without enzyme water, mix; and so on. Finally, 1ul of each tube is taken as a template of the fluorescent quantitative PCR.
The Q-PCR results were as follows:
the plotted standard graph is shown in fig. 2.
2.3 RNA reverse transcription PCR reaction
The concentration of the extracted total RNA is determined, then RNA is quantified, and cDNA is synthesized from the 3' end according to the set reverse transcription program.
1. The following reagents were added to a sterile, enzyme-free tube in order:
2. mixing gently, centrifuging instantly, and incubating at 65 deg.C for 5 min;
3. adding the following reagents into the mixed solution, wherein the final volume is 20 ul;
4. gently mix and centrifuge instantaneously, operating according to the following procedure:
the cDNA (complementary DNA) obtained after completion of the reaction was stored at-20 ℃.
2.4 real-time fluorescent quantitative PCR
SYBR method Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, q-PCR) reaction system:
iTaqTMUniversal
Green Supermix：
SYBR method real-time fluorescence quantitative PCR reaction steps:
after the reaction, the amplification curve and the melting curve of the real-time fluorescent quantitative PCR were confirmed, and the expression intensity of each gene was normalized based on the CT value (threshold cycle values) and the internal reference gene (pGL3), and the P value was calculated by unpaired t-test.
The real-time fluorescent quantitative PCR reaction system of the Taqman probe method comprises the following steps:
iTaqTMUniversal Probes Supermix：
taqman probe Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, q-PCR) reaction system:
the Taqman probe real-time fluorescence quantitative PCR reaction step:
after completion of the reaction, the expression intensity of each gene was confirmed by labeling with CT value (threshold cycle values) and reference gene (pGL3), and then calculating P value by unpaired t-test.
2.5 statistical analysis
Statistical analysis was performed using SPSS 13.0 and Graphpad 5.0 software.
3 results
3.1 LOC284454, circMAN1A2 and circRNF13 are highly expressed in serum of patients with nasopharyngeal carcinoma
The experiment adopts 100 nasopharyngeal carcinoma patient serums and 51 normal control serums, and the experimental result shows that: LOC284454, circMAN1A2 and circRNF13 were up-regulated in serum from patients with nasopharyngeal carcinoma compared to normal controls, see FIG. 3.
In order to ensure the authenticity and reliability of experimental data and reduce errors in the experimental process, Taqman probes of LOC284454, circMAN1A2 and circRNF13 are designed, the expression of LOC284454, circMAN1A2 and circRNF13 in the serum of a nasopharyngeal carcinoma patient is detected by adopting an experimental method of Taqman probe q-PCR, and a normal control is increased to 121 cases, as shown in figure 4. The results show that: compared with the serum of a normal control group, the expression levels of LOC284454, circMAN1A2 and circRNF13 in the serum of a nasopharyngeal carcinoma patient are obviously increased, and the statistical significance is realized (P is less than 0.001).
3.2 nasopharyngeal carcinoma ROC curve evaluation clinical application value
The invention verifies that the expression of LOC284454, circMAN1A2 and circRNF13 is up-regulated in nasopharyngeal carcinoma, and the clinical application values of LOC284454, circMAN1A2 and circRNF13 are evaluated by adopting an ROC curve.
When the AUC value is between 0.5 and 1.0, and the AUC is greater than 0.5, the closer the AUC is to 1, the better the specificity and the sensitivity are, and the higher the clinical application value is.
We performed ROC curve analysis based on the expression levels of LOC284454, circMAN1a2 and circRNF13 in nasopharyngeal carcinoma, as shown in figure 5. ROC curve analysis LOC284454 gave AUC values of 0.931; the AUC value for circMAN1a2 was 0.911; the AUC value of circRNF13 was 0.877; the AUC value of ROC curve analysis of the combination of the three molecules is 0.968, which indicates that the combination of the three molecules has higher accuracy in tumor diagnosis.
LOC284454 Single molecule ROC Curve analysis
Area under the curve (AUC)
Analyzed parameter LOC284454
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
Circman1A2 Single molecule ROC Curve analysis
Area under the curve (AUC)
Parameter analyzed as circMAN1A2
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
Combining three molecular ROC curve analyses of LOC284454, circRNF13 and circMAN1A2
Area under the curve (AUC)
Analyzed parameters LOC284454, circRNF13 and circMAN1A2
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
3.3 LOC284454, circMAN1A2 and circRNF13 are highly expressed in the serum of patients with oral cancer
The experiment adopts 55 cases of oral cancer patient serum and 121 cases of normal control serum, designs Taqman probes of LOC284454, circMAN1A2 and circRNF13, adopts an experimental method of Taqman probe q-PCR to detect the expression of LOC284454, circMAN1A2 and circRNF13 in the oral cancer patient serum, and is shown in figure 6. The results show that: compared with the serum of a normal control group, the expression levels of LOC284454, circMAN1A2 and circRNF13 in the serum of oral cancer patients are obviously increased, and the statistical significance is achieved (P is less than 0.001).
3.4 oral cancer ROC Curve assessment clinical application value
The invention verifies that the expression of LOC284454, circMAN1A2 and circRNF13 is up-regulated in oral cancer, and the clinical application values of LOC284454, circMAN1A2 and circRNF13 are evaluated by adopting an ROC curve.
We performed ROC curve analysis based on the expression levels of LOC284454, circMAN1a2 and circRNF13 in oral cancer, as shown in figure 7. ROC curve analysis LOC284454 gave an AUC value of 0.698; the AUC value for circMAN1a2 was 0.779 and the AUC value for circRNF13 was 0.731; the AUC value of ROC curve analysis of the combination of the three molecules is 0.863, which indicates that the combination of the three molecules has higher accuracy in tumor diagnosis.
IncLOC284454 Single molecule ROC Curve analysis
Area under the curve (AUC)
Analyzed parameter LOC284454
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
circMAN1A2 single molecule ROC curve analysis
Area under the curve (AUC)
Parameter analyzed as circMAN1A2
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
Combining three molecular ROC curve analyses of LOC284454, circRNF13 and circMAN1A2
Area under the curve (AUC)
Analyzed parameters LOC284454, circRNF13 and circMAN1A2
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
3.5 LOC284454, circMAN1A2 and circRNF13 are highly expressed in the serum of thyroid cancer patients
In the serum of 57 cases of thyroid cancer patients and the serum of 121 cases of normal controls, the expression level of LOC284454, circMAN1A2 and circRNF13 in the serum of thyroid cancer patients is detected by adopting an experimental method of Taqman probe q-PCR, and the result shows that: LOC284454, circMAN1A2 and circRNF13 were significantly elevated in serum from thyroid cancer patients compared to normal controls, and were statistically significant (P < 0.001), as shown in FIG. 8.
3.6 thyroid cancer ROC curve evaluation clinical application value
The invention verifies that the expression of LOC284454, circMAN1A2 and circRNF13 is up-regulated in thyroid cancer, and the clinical application value of LOC284454, circMAN1A2 and circRNF13 is evaluated by adopting an ROC curve.
We performed ROC curve analysis based on the expression levels of LOC284454, circMAN1a2 and circRNF13 in thyroid cancer, as shown in figure 9. ROC curve analysis LOC284454 gave an AUC value of 0.834; the AUC value for circMAN1a2 was 0.734 and the AUC value for circRNF13 was 0.798; the AUC value of the ROC curve analysis of the combination of the three molecules is 0.881, which indicates that the accuracy of the tumor diagnosis of the combination of the three molecules is higher.
LOC284454 Single molecule ROC Curve analysis
Area under the curve (AUC)
Analyzed parameter LOC284454
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
circMAN1A2 single molecule ROC curve analysis
Area under the curve (AUC)
Parameter analyzed as circMAN1A2
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
Single molecule ROC curve analysis of circRNF13
Area under the curve (AUC)
Parameter analyzed as circRNF13
a. Based on nonparametric assumptions
b. Zero hypothesis true area is 0.5
Combining three molecular ROC curve analyses of LOC284454, circRNF13 and circMAN1A2
Area under the curve (AUC)
Analyzed parameters LOC284454, circRNF13 and circMAN1A2
a. Based on nonparametric assumptions
b. Zero assumes that the real area is 0.5.
Sequence listing
<110> university of south-middle school
Application of <120> reagent for detecting three non-coding RNAs and kit
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 553
<212> DNA
<213> Unknown (Unknown)
<400> 1
ggaagaggaa gaacgtctga gaaataaaat tcgagctgat catgagaagg ccttggaaga 60
agcaaaagaa aaattaagaa agtcaagaga ggaaattcga gcagaaattc agacagagaa 120
aaataaggta gtccaagaaa tgaagataaa agagaacaag ccactgccac cagtccctat 180
tcccaacctt gtaggaatac gtggtggaga cccagaagat aatgacataa gagagaaaag 240
ggaaaaaatt aaagagatga tgaaacatgc ttgggataac tataggacat atgggtgggg 300
acataatgaa ctcagaccta ttgcaaggaa aggacactcc cctaacatat ttggaagttc 360
acaaatgggt gctaccatag tagatgcttt ggataccctt tatatcatgg gacttcatga 420
tgaattccta gatgggcaaa gatggattga agacaacctt gatttcagtg tgaattcaga 480
ggtgtctgtg tttgaagtca acattcgatt tattggaggc ctacttgcag catattacct 540
<210> 2
<211> 716
<212> DNA
<213> Unknown (Unknown)
<400> 2
gtgattttac aacgagatgc tgctctccat agggatgctc atgctgtcag ccacacaagt 60
ctacaccatc ttgactgtcc agctctttgc attcttaaac ctactgcctg tagaagcaga 120
cattttagca tataactttg aaaatgcatc tcagacattt gatgacctcc ctgcaagatt 180
tggttataga cttccagctg aaggtttaaa gggttttttg attaactcaa aaccagagaa 240
tgcctgtgaa cccatagtgc ctccaccagt aaaagacaat tcatctggca ctttcatcgt 300
gttaattaga agacttgatt gtaattttga tataaaggtt ttaaatgcac agagagcagg 360
atacaaggca gccatagttc acaatgttga ttctgatgac ctcattagca tgggatccaa 420
cgacattgag gtactaaaga aaattgacat tccatctgtc tttattggtg aatcatcagc 480
taattctctg aaagatgaat tcacatatga aaaagggggc caccttatct tagttccaga 540
atttagtctt cctttggaat actacctaat tcccttcctt atcatagtgg gcatctgtct 600
catcttgata gtcattttca tgatcacaaa atttgtccag gatagacata gagctagaag 660
aaacagactt cgtaaagatc aacttaagaa acttcctgta cataaattca agaaag 716
<210> 3
<211> 1774
<212> RNA
<213> Unknown (Unknown)
<400> 3
ggggucaagc ccccuuggag ccugcagccc cugccuuccc ugggugggcu gaugcuugga 60
gcagagauga ggacucagaa ucagaccugu gucuggagga gggauguggu gggugggguu 120
ggcugggccc aaaugugugc ugcaggcccu gauccccaac ucugcaacug gggaccccug 180
cauggccaca gcucaggcug ggcuguggug ccagcauaga uaggugggug aguggguggc 240
ccuuccauua aaagggaagc cagcuguguc cuuuccgggc cuggaggcuu ggccccuccu 300
cucccaagcc uggcaggggc acuggcccgg cccgcaccuu ccuagcagcc aguuacccaa 360
gaggaagcug ccuugggccu ccagaccguu aaaugccaac uccuggcuuc cgguaucagg 420
cuggguugac cugaccuggc cccuucuugc ugggcccugc agcuuucuaa cuugccggga 480
ggagcaguga cacccgcccc acaugugggg cauggaacaa guuccuugug gacccagaag 540
ggacacaagc aggugugcuu aguccugagg cgcugggaau agcugauccu cccugccuug 600
aggggguucu cagggcaggg aagaguuagg acucuguuuu uuuuuuuuug uuuuuuuuuu 660
uuugagaugg agucucgcac ugucacccgg gcuggagugc aauggcucga ucucggcuca 720
cugcaaccuc caccucccca guucacacga uucuccugcc ucagccuccc aaguagcugg 780
gauuacaggu gcacaccacc gcaccuggcu aauuuuugua uuuuuaguag agaccgaguu 840
ucgccauguu ggccaggcug gucucgaacu cuugaccuca ggugaucugc ccgccucagc 900
cacccaaagu guugggauua caggcaugag ccacugcgcc cggccaauuu uuuuuuaugu 960
uuuguagaga cgggguuucg ccauguuacc cagacugguc uugaacuccu gaccucaagc 1020
aaucugcucg ucuuagccuc ccaaagugcu gggauuacag cugugagcca ccgugccugg 1080
ccuuuuauug uuuguuuuug agacggaguc ucacucuguu gcccaggcug aagugcagug 1140
gugugaucuu ggcucacugc aaccucugcc uccuggguuc aagcgauucu ccugccucag 1200
ccuccugagu agcugggcuu acaggcaccc accaccaugc ccggcuaauc uuuguauuuu 1260
uaguagagac gggguuucac caucuuggcc aggcuggugu gaucauggcu cauugcaacc 1320
uugaauuccu gggcacaagu gauccuccug ccuuagccuc cccaguagag cugggacuac 1380
agguaugcgc caccacaccu ggcuaauuuu uuuaauuuua auuuuuguag agaugggggg 1440
gcaggucuca cuauguugcc caggcuguuc ucgaacuccu ggccacaagc cauccuccca 1500
ccuuagucuc ccaaugcgcc ggaauuacag guggcucagg ugugagccac cgugccuggc 1560
uuuucuccac uaucuugaaa ucagauggga ggaggcuuuu uucugggugg gacugaggag 1620
gcacacugaa gucccccagg ucaucggggc ugggccauug ccuuuuuccc cacccugggu 1680
agucguggac agaagcuugg gaugggaugg agaggagaga ucgugcugug ugucaugucu 1740
guuguucaag uaaauaaaag uugcccugac uuca 1774
<210> 4
<211> 21
<212> DNA
<213> Unknown (Unknown)
<400> 4
agatgggcaa agatggattg a 21
<210> 5
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 6
<210> 7
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 7
<210> 8
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 9
<210> 10
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 10
<210> 11
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 11
<210> 12
<211> 36
<212> DNA
<213> Unknown (Unknown)
<400> 12
caaagatgga ttgaagacaa ccttgatttc agtgtg 36
<210> 13
<211> 35
<212> DNA
<213> Unknown (Unknown)
<400> 13
aggatagaca tagagctaga agaaacagac ttcgt 35
<210> 14
<211> 27
<212> DNA
<213> Unknown (Unknown)
<400> 14
cgtgcctggc ttttctccac tatcttg 27
<210> 15
<211> 22
<212> DNA
<213> Unknown (Unknown)
<400> 15
acgcaggtgt cgcaggtctt cc 22
Claims (3)
1. The application of the reagent for detecting three non-coding RNAs is characterized in that the reagent is used for preparing a tumor auxiliary diagnostic preparation, the three non-coding RNAs are circular RNA circMAN1A2, the sequence of the circular RNA circRNF13 is shown as SEQ No.1, the sequence of the circular RNA circRNF13 is shown as SEQ No.2, and the sequence of the long-chain non-coding RNA LOC284454 is shown as SEQ No. 3; the three non-coding RNA detection reagents are used for detecting three non-coding RNAs in serum; tumor types include: nasopharyngeal carcinoma and thyroid cancer.
2. The use of claim 1, wherein the detection of three non-coding RNA agents comprises a real-time fluorescent quantitative detection agent.
3. The use of claim 2, wherein the real-time fluorescence quantitative detection reagent comprises primers for real-time fluorescence quantitative detection of circular RNA circMAN1A2 expression:
an upstream primer: 5'-AGATGGGCAAAGATGGATTGA-3'
A downstream primer: 5'-GCCTTCTCATGATCAGCTCG-3', respectively;
and a primer for real-time fluorescent quantitative detection of circular RNA circRNF13 expression:
an upstream primer: 5'-GTCCAGGATAGACATAGAGC-3'
A downstream primer: 5'-GTGTAGACTTGTGTGGCTGA-3', respectively;
and a primer for real-time fluorescent quantitative detection of long-chain non-coding RNA LOC284454 expression:
an upstream primer: 5'-ATTACAGGTGGCTCAGGTGT-3'
A downstream primer: 5'-CTTCAGTGTGCCTCCTCAGT-3' are provided.
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Non-Patent Citations (2)
| Title |
|---|
| CircRNF13 regulates the invasion and metastasis in lung adenocarcinoma by targeting miR-93-5p;Wang et al.;《Gene》;20180425;摘要，2. Materials and methods * |
| Heterogeneous circRNA expression profiles and regulatory functions among HEK293T single cells;Zhong C.F.等;《Scientific Reports》;20171031;Discussions、第1页 * |
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