CN108660213A - The application of three kinds of non-coding RNA reagents of detection and kit - Google Patents
The application of three kinds of non-coding RNA reagents of detection and kit Download PDFInfo
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Abstract
The invention discloses the application of three kinds of non-coding RNA reagents of detection and kits.The especially preparation of the Real time PCR method auxiliary diagnosis tumour of detection circular rna circMAN1A2, circular rna circRNF13, long-chain non-coding RNA LOC284454.CircMAN1A2, circRNF13, LOC284454 equal up-regulated expression in nasopharyngeal carcinoma, carcinoma of mouth, thyroid cancer patients serum are confirmed by research.Therefore, the auxiliary diagnosis by the expression joint of circMAN1A2, circRNF13, LOC284454 for tumour, further promotes the accuracy of diagnosis, has far-reaching clinical meaning and important popularizing application prospect.
Description
Technical field
The invention belongs to oncomolecularbiology technical fields, and in particular to a kind of detection circular rna
The reagent of circMAN1A2, circular rna circRNF13 and long-chain non-coding RNA LOC284454 are preparing tumour
Application on auxiliary diagnosis preparation and corresponding kit.
Background technology
Malignant tumour seriously endangers the health of the mankind, and the trend risen is presented in China in Recent Years Cancer Mortality.
According to Cancer in China statistics center statistics, malignant tumour neopathy number is about 380.4 ten thousand, wherein male patient 211.4 ten thousand, female
Property patient 169.0 ten thousand, about every day, which is more than 10,000 person-times, can be diagnosed as cancer, every about point just having 7 person-times to be diagnosed as
Cancer.Cancer Mortality is 278.07/10 ten thousand, and wherein the incidence of male malignancy is 301.67/10 ten thousand, and women dislikes
Property tumor incidence be 253.29/10 ten thousand.The win bit rate of Cancer Mortality is that 190.63/10 ten thousand (population standard rate is pressed
Chinese standard population structure in 2000), Cancer Mortality generation mark rate is that 186.53/10 ten thousand (population standard rate is pressed
Segi's world standards population structure), 0-74 Sui cumulative morbidity is about 21.58%.Mortality of malignant tumors is about
167.89/10 ten thousand, wherein win bit rate about 106.98/10 ten thousand, the cumulative mortality of generation mark rate about 106.09/10 ten thousand, 0-74 Sui is about
It is 12.00%.
It is counted according to American Cancer Society (ACS), the estimated neopathy number about 173.5 ten thousand of U.S.'s malignant tumour, average daily new hair
4700.The incidence of U.S.'s malignant tumour declines steadily, while mortality of malignant tumors is declined with 1.5% speed every year.
U.S.'s Cancer Mortality is decreased obviously, and may be attributed to control smoking, be promoted multi-level screening for cancer and cancer is novel
The extensive use of therapy.
It can be seen that reducing the pathogenic factors of malignant tumour, effective early diagnosis and the spy of novel cancer therapies
Rope plays the role of the control of cancer great.So being just dedicated to finding novel tumor-marker in the medical research in China
Object, to the early diagnosis for malignant tumour, and facilitate as early as possible accurately make a definite diagnosis the state of an illness to be conducive to treatment provide it is new
Approach.
Circular rna (circual RNA, circRNA) is a new class of RNA molecule, configuration and traditional linear rna
(containing 5 ' and 3 ' ends) is different, and circular RNA molecule is in closed circular structure (i.e. 5 ' and 3 ' ends join end to end), due to not dissociating
5 ' and 3 ' ends, therefore circular rna than linear rna more stablize.Originally circRNA is considered as a kind of pair of splicing mistake
Product, and it is not affected by extensive concern, in recent years, the RNA-Sequencing based on new-generation sequencing technology is extensively using promotion
The rapid development in many fields RNA, a large amount of new circRNA are found, to which circRNA has been pushed to malignant tumour gene
Group learns the stage center of research.
CircRNA as a kind of emerging RNA, row at and functional mechanism attract more and more researchs.With
More and more circRNA are authenticated, it is found that the expression of some circRNA is significantly larger than the table of its dependent linearity gene
It reaches.CircRNA forms cyclic structure with covalent bond, and most circRNA are the products reversely spliced, are widely expressed in body,
Sometimes its expression is 10 times of linear rna, is positioned at cytoplasm mostly.CircRNA can be inhaled as the molecule sponge of miRNA
Attached miRNA, conjugated protein play important biological function as regulatory factor of genetic transcription and protein translation etc.,
The very possible biomarker and clinical treatment target new as malignant tumour, new think of is provided for the diagnosis and prognosis of tumour
Road.
Long-chain non-coding RNAs (long non-coding RNAs, lncRNAs) are that a kind of length is more than 200nt, lacks
The RNAs molecules of special complete open reading frame, nothing or few encoding histone functions.It has recently been demonstrated that lncRNAs
By adjusting the expression of gene extensively in epigenetic, transcription and post-transcriptional level, they take part in biology growing, development,
The regulation and control of the important vital movement such as aging and death.More and more research shows that the unconventionality expression or afunction of lncRNAs
It is closely related with the occurrence and development of tumour.Some lncRNAs molecules have important meaning in terms of the diagnosing and treating of tumour
Justice, and new molecular labeling can be judged as tumor prognosis.
Therefore, it is necessary to screen and verify more biomarkers of the novel non-coding RNA as cancer diagnosis and prognosis
And its application, and the protection that can be got well as early as possible in patent field, international competition of the China in the technical field can be obviously improved
Power.
Invention content
The present invention filters out the circRNA and IncRNA of high expression from RNA seq data, and respectively a plurality of types of
Their expression is successively verified in cancer, diagnosing malignant tumor can be used as by searching out emerging circRNA and IncRNA
Serum molecules marker.
Therefore, the first purpose of the invention is to provide a kind of application of three kinds of non-coding RNA reagents of detection, the examinations
Agent is used to prepare tumour auxiliary diagnosis preparation, and three kinds of non-coding RNAs are circular rna circMAN1A2, sequence such as SEQ No.1
Shown, circular rna circRNF13, sequence is as shown in SEQ No.2, and long-chain non-coding RNA LOC284454, sequence is such as
Shown in SEQ No.3.For tumour auxiliary diagnosis provide it is a kind of it is new accurately and reliably, easy detection approach.
Serum is as detection sample, and compared to tissue detection, sample acquisition mode and easy to operate is at low cost, accurately
Property it is high, the diagnosis for tumour is ideal.Therefore, applicant uses serum as sample in the course of the research, by a large amount of
Experiment find and verification serum in the relevant molecule of diagnosing tumor and their existing relationships between tumour.Applicant
The circular rna circMAN1A2 present in serum, circular rna circRNF13 and long-chain non-coding RNA are found for the first time
There are positively related relationships with a plurality of types of tumours by LOC284454, by detecting its content in serum, it may be convenient to
For assist or tentative diagnosis suffer from tumour possibility.
The present invention is using the experimental method of SYBR-qPCR Taqman probe q-PCR respectively in nasopharyngeal carcinoma, oral cavity
The expression of circMAN1A2 and circRNF13 and LOC284454 are successively verified in cancer, thyroid cancer.
Since the present invention is for verifying sample collected by the expression of circMAN1A2, circRNF13, LOC284454
This number is sufficient, has statistical significance, and samples sources are reasonable, and screening criteria is stringent, and experiment process is rigorous, and real result can
It leans on.
Test result of the present invention shows circMAN1A2, circRNF13, LOC284454 in nasopharyngeal carcinoma, carcinoma of mouth, first shape
Apparent up-regulation is expressed in serum of adenocarcinoma patients, is showed the consistency for expressing trend in multiple types tumour, is implied
Circ MAN1A2, circRNF13, LOC284454 molecule can be individually as the molecular marked compounds of diagnosing tumor, but pass through
Statistical analysis discovery is crossed, three kinds of non-coding RNAs join together to judge jointly, accuracy higher.
In above application, the reagent of three kinds of non-coding RNAs of detection includes real time fluorescent quantitative detection reagent.
In order to ensure the specificity of primer and the accuracy of qPCR, applicant follows strictly RNA design of primers principles, designs
Go out the primer that can accurately expand for real time fluorescent quantitative detection circular rna MAN1A2 expression：
Sense primer：5’-AGATGGGCAAAGATGGATTGA-3’
Downstream primer：5’-GCCTTCTCATGATCAGCTCG-3’；
And the primer for real time fluorescent quantitative detection circular rna RNF13 expression：
Sense primer：5’-GTCCAGGATAGACATAGAGC-3’
Downstream primer：5’-GTGTAGACTTGTGTGGCTGA-3’.
And the primer for real time fluorescent quantitative detection long-chain non-coding RNA LOC284454 expression：
Sense primer：5’-ATTACAGGTGGCTCAGGTGT-3’
Downstream primer：5’-CTTCAGTGTGCCTCCTCAGT-3’
Second object of the present invention is to provide a kind of kit for tumour auxiliary diagnosis；It is carried for tumour auxiliary diagnosis
A kind of new accurately and reliably detection product is supplied.
The kit, which contains, can detect circular rna circMAN1A2 in serum, circular rna circRNF13, and
The reagent of long-chain non-coding RNA LOC284454；The sequence of the circular rna circMAN1A2 is as shown in SEQ No.1, institute
The sequence of the circular rna circRNF13 stated is as shown in SEQ No.2, the sequence of the long-chain non-coding RNA LOC284454
Row are as shown in SEQ No.3.
Further, the preferably following primer of the primer that circular rna circMAN1A2 can be expanded：
Sense primer：5’-AGATGGGCAAAGATGGATTGA-3’
Downstream primer：5’-GCCTTCTCATGATCAGCTCG-3’.
And the preferably following primer of primer of circular rna circRNF13 can be expanded：
Sense primer：5’-GTCCAGGATAGACATAGAGC-3’
Downstream primer：5’-GTGTAGACTTGTGTGGCTGA-3’.
The primer of long-chain non-coding RNA LOC284454 can be expanded：
Sense primer：5’-ATTACAGGTGGCTCAGGTGT-3’
Downstream primer：5’-CTTCAGTGTGCCTCCTCAGT-3’
But it is of the present invention to expand circular rna circMAN1A2 and circRNF13 and long-chain non-coding RNA
The primer of LOC284454 is not limited to the primer of above-mentioned offer.
Since, without reference gene, we have been preferably added to PGL3 plasmids as reference, to reduce RNA extractings in human serum
Error in the process, for example, RNA adsorption efficiencies, elution efficiency difference, while also for research relatively reliable experiment number is provided
According to.Because of the GL3 plasmids being quantitatively adding, so that it may it is corresponding to calculate target gene with the ct values according to q-PCR reaction gained
Copy number, so as to reach absolute quantitation.It is outer ginseng gene addition, reduce experiment present in systematic error, to for
Research provides the higher data information of confidence level.
Therefore,
Further, kit of the present invention further includes：Internal reference pGL3 primers：
Sense primer：5’-TCCATCTTGCTCCAACACCC-3’
Downstream primer：5’-TCGTCTTTCCGTGCTCCAAA-3’.
But the internal control primer of the present invention is not limited only to the internal reference pGL3 primers of above-mentioned offer.
Specificity of the Taqman probes with height, high sensitivity and specificity are preferably.SYBR methods need to manage in different EP
Middle progress PCR reactions, and Taqman sonde methods may be implemented in an EP pipe while detect multiple genes, to reduce again
Experimental error.The experimental method of Taqman probe-qPCR is more rigorous, and reliability and repeatability are higher, and can be with a variety of samples
This is detected simultaneously, the favorable repeatability of experiment, and the obtained data of experiment are more reliable, provided for the judge of clinical value
Sturdy basis.
Therefore,
Further, kit of the present invention preferably further includes：Circular rna is detected for real time fluorescent quantitative
The mating Taqman probes of primer of circMAN1A2 expression：5’-ROX-
CAAAGATGGATTGAAGACAACCTTGATTTCAGTGTG-BHQ2-3’。
And the Taqman probes that primer for real time fluorescent quantitative detection circular rna circRNF13 expression is mating：
5’-Cy5-AGGATAGACATAGAGCTAGAAGAAACAGACTTCGT-BHQ2 -3’。
The mating Taqman spies of the primer of long-chain non-coding RNA LOC284454 expression are detected for real time fluorescent quantitative
Needle：
5’-FAM–CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3’
Further, kit of the present invention preferably further includes：The mating Taqman probes of internal reference pGL3 primers：
5’-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1-3’。
But Taqman probes used in the present invention are not limited only to the partner probe of above-mentioned offer.
Further, also contain in kit of the present invention：Serum total RNA extraction reagent, RNA reverse transcription PCR reaction reagents
With real time fluorescent quantitative detection reagent.
The present invention is fixed from reverse transcription, real-time fluorescence after extracting RNA in kinds of tumors patients serum and normal human serum sample
Amount method has detected the expression of circMAN1A2 and circRNF13, LOC284454, as a result shows circMAN1A2, circRNF13
With LOC284454 in a plurality of types of Serum of Cancer Patients equal up-regulated expression.Find that there are circular rnas point in serum for the first time
There is unification with a plurality of types of tumours in sub- circMAN1A2 and circRNF13 and long-chain non-coding RNA LOC284454
Positive correlativity, to prompt circMAN1A2, circRNF13, LOC284454 to can be used as the molecule mark of tumour auxiliary diagnosis
Will, especially triple combination are got up, accuracy rate higher.The present invention provides strong molecular biosciences for the auxiliary diagnosis of tumour
Tool has far-reaching clinical meaning and important popularizing application prospect.
Description of the drawings
Fig. 1 is circMAN1A2 and circRNF13 collection of illustrative plates；
A is circMAN1A2, b circRNF13 in figure.
Fig. 2 is internal reference plasmid pGL3 Q-PCR standard curves.
Fig. 3 is that SYBR-qPCR detects LOC284454, circRNF13, circMAN1A2 in patients with nasopharyngeal carcinoma
Expression；
A in figure, b, c are respectively that SYBR-qPCR detection LOC284454, circRNF13, circMAN1A2 suffers from nasopharyngeal carcinoma
Expression in person's serum, wherein compared with Normal group, LOC284454, circRNF13, circMAN1A2 are in nasopharynx
Up-regulated expression in cancer patients serum, P ＜ 0.001；N represents Normal group, and T represents nasopharyngeal carcinoma group, n representative sample numbers.*P<
0.05,**P<0.01,***P<0.001。
Fig. 4 is that Taqman-qPCR detects LOC284454, circRNF13 and circMAN1A2 in patients with nasopharyngeal carcinoma
Expression；
A in figure, b, c are respectively that Taqman-qPCR detects LOC284454, circRNF13 and circMAN1A2 in nasopharynx
Expression in cancer patients serum, wherein compared with Normal group, LOC284454, circRNF13 and circMAN1A2
The up-regulated expression in patients with nasopharyngeal carcinoma, P ＜ 0.001；N represents Normal group, and T represents nasopharyngeal carcinoma group, n representative samples
Number.*P<0.05,**P<0.01,***P<0.001.
Fig. 5 is nasopharyngeal carcinoma group ROC curve；
A in figure, b, c, d are respectively LOC284454, circRNF13, circMAN1A2, LOC284454 joint
The ROC curve of circMAN1A2 and circRNF13.
Fig. 6 is that Taqman-qPCR detects LOC284454, circRNF13 and circMAN1A2 in oral cancer patient's serum
Expression；
A in figure, b, c are respectively that Taqman-qPCR detects LOC284454, circRNF13 and circMAN1A2 in oral cavity
Expression in cancer patients serum, wherein compared with Normal group, LOC284454, circRNF13 and circMAN1A2
The up-regulated expression in oral cancer patient's serum, P ＜ 0.001；N represents Normal group, and T represents carcinoma of mouth group, n representative samples
Number.*P<0.05,**P<0.01,***P<0.001.
Fig. 7 is carcinoma of mouth group ROC curve；
A in figure, b, c, d are respectively LOC284454, circRNF13, circMAN1A2, LOC284454 joint
CircMAN1A2 and the united ROC curves of circRNF13.
Fig. 8 is that Taqman-qPCR detects LOC284454, circRNF13 and circMAN1A2 in thyroid cancer patients serum
In expression；
A in figure, b, c are respectively that Taqman-qPCR detects LOC284454, circRNF13 and circMAN1A2 in first shape
Expression in serum of adenocarcinoma patients, wherein compared with Normal group, LOC284454, circRNF13 and
CircMAN1A2 up-regulated expressions in thyroid cancer patients serum, P ＜ 0.001；N represents Normal group, and T represents thyroid cancer
Group, n representative sample numbers；*P<0.05,**P<0.01,***P<0.001.
Fig. 9 is thyroid carcinoma group ROC curve；
A in figure, b, c, d are respectively LOC284454, circRNF13, circMAN1A2, LOC284454 joint
CircMAN1A2 and the united ROC curves of circRNF13.
Specific implementation mode
It is intended to further illustrate the present invention below in conjunction with specific implementation mode, and non-formation limitation of the present invention.
The Normal group used in the present invention and tumor group serum sample, are respectively from Xiangye No. 2 Hospital of Central South University
Medical center and Hunan Provincial Tumour Hospital inspection center.Normal group collects 121 altogether, has excluded tumor disease, without biography
It catches an illness, serious immunity disease and other major diseases；Tumor group serum sample includes：100, patients with nasopharyngeal carcinoma sample,
Oral cancer patient's serum sample sheet 55, thyroid cancer patients serum sample 57, blood sample collection time interval：In March, 2017 is extremely
In January, 2018.The blood sample of collection gradually complete clinical data, including patient's name, gender, age, be hospitalized number, pathology class
Type, identification card number, receives treatment etc. at pathological staging.All serum samples obtain the agreement of gathered person or patient, by
Step establishes the more complete blood sample library of clinical data.
1, Realtime PCR primers
Primer and probe used in the present invention is designed by dedicated design of primers website Primer 3.0.Primer
Synthetic work entrusts the synthesis of Qing Ke bio-engineering corporations.In order to ensure the specificity of primer and the accuracy of qPCR, we are stringent
Follow following design of primers principle：
Probe design principle：
Primer and Taqman probe sequence used herein is as follows：
(1) pGL3 primers
Sense primer：5’-TCCATCTTGCTCCAACACCC-3’；
Downstream primer：5’-TCGTCTTTCCGTGCTCCAAA-3’；
Taqman probes：5’-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1-3’；
(2) LOC284454 primers
Sense primer：5’-ATTACAGGTGGCTCAGGTGT-3’
Downstream primer：5’-CTTCAGTGTGCCTCCTCAGT-3’
Taqman probes
5’-FAM–CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-3’
(3) circMAN1A2 primers
Sense primer：5’-AGATGGGCAAAGATGGATTGA-3’；
Downstream primer：5’-GCCTTCTCATGATCAGCTCG-3’；
Taqman probes：5’-ROX-
CAAAGATGGATTGAAGACAACCTTGATTTCAGTGTG-BHQ2-3’；
(4) circRNF13 primers
Sense primer：5’-GTCCAGGATAGACATAGAGC-3’
Downstream primer：5’-GTGTAGACTTGTGTGGCTGA-3’
Taqman probes：5’-Cy5-AGGATAGACATAGAGCTAGAAGAAACAGACTTCGT -BHQ2-3’；
CircMAN1A2 and CircRNF13, is shown in Fig. 1.
2 methods
2.1 cancer patient's peripheral blood samples acquire
Sample collection flow is as follows：
(1) it uses EDTA anticoagulant blood-collecting pipes to collect periphery blood specimen, acquires 1~2ml of limosis vein blood, blood sampling of turning upside down
Soft mixing is managed, haemolysis is avoided；
(2) by 4 DEG C of whole blood sample, 1600g centrifuges 10min, and extraction 500-1000 μ l blood plasma is placed in the EP pipes of precooling,
Patient's name and patient's identification number or the name of normal person are indicated on pipe；
(3) -80 DEG C of refrigerators are placed after plasma specimen extraction immediately to preserve, whole flows are after blood is in vitro in 4 hours
Reason finishes；
(4) it fills in《Sample information record sheet》, collect the information such as patient and normal person's name, gender, age and patient live
Institute's number, pathological staging, identification card number, receives the clinical datas such as treatment at histological type.
2.2 serum total serum IgEs extract
Using miRNeasy Serum/PlasmaKit kits, it is as follows：
(1) 200ul blood plasma is placed in 2ml EP pipes；
(2) lysate (i.e. 1000ul) of 5 times of volumes is added to EP pipes, is vortexed or is mixed well with liquid-transfering gun；
(3) by (15-25 DEG C) placement 5min of lysate room temperature in EP pipes；
(4) outer ginseng PGL3 is added and mixes well (1.6*108copies/ul)；
(5) chloroform (i.e. 200ul) of equal samples volume is added, is vortexed or exerts oneself to shake 15s；
(6) (15-25 DEG C) placement 2-3min of room temperature；
(7) 12000g is centrifuged, 4 DEG C, 15min, is finished debugging centrifuge to room temperature, upper layer colourless aqueous phase is RNA；Middle level is white
Color is DNA；Lower layer's red organic phase is albumen；
(8) it draws in upper strata aqueous phase to new 2ml EP pipes (600ul), the alcohol of 1.5 times of volumes 100% is added
900ul is mixed well with liquid-transfering gun, and alcohol is added and is likely to form precipitation but without influence；
(9) pillar is put into 2ml tube pipes, draws 700ul to pillar, fastens lid centrifugation >=8000g
(10000rpm), 15s, room temperature (15-25 DEG C) abandon waste liquid in collecting pipe；
(10) (9) are repeated, remaining sample is crossed into pillar, abandons waste liquid in collecting pipe；
(11) 700ul Buffer RWT are added to pillar, fasten lid centrifugation, >=8000g (10000rpm), 15s are abandoned
Waste liquid in collecting pipe；
(12) 500ul Buffer RPE are added to pillar, fasten lid centrifugation, >=8000g (10000rpm), 15s are abandoned
Waste liquid in collecting pipe；
(13) 80% alcohol of 500ul is added in pillar, gently fastens lid centrifugation, >=8000g (10000rpm), 2min are washed
Pillar abandons collecting pipe (carefully moving pillar, avoid the waste liquid for being stained with collecting pipe)；
(14) pillar is put into new 2ml Tube pipes, opens lid, centrifuge 5min at full speed, it is dry, abandon collecting pipe (for
Damage lid is avoided, is spaced apart, keeps lid opposite with rotor steering upward)；
(15) pillar is put in new 1.5ml Tube pipes, 14ul is added without enzyme water to pillar film center, gently closes column
Son centrifuges 1min at full speed, about 12ul in eluted rna to 1.5ml Tube pipes.
We add PGL3 plasmids as reference, and relatively reliable experimental data is provided for the present invention.
Addition is to add pGL3 about 2 × 10 per ml serum with reference to plasmid concentration8Copy about adds per ml serum according to calculating
1ng plasmids；
The 10 μ l reaction systems that Real time PCR are used are as follows：
PGL3 plasmid control Drawing of Curve processes are as follows：It takes 1ng plasmids to add no enzyme water constant volume to 500 μ l, then therefrom takes 2 μ l
Add no enzyme water constant volume to 1ml, as the 1st pipe；Then gradually half-and-half dilute, that is,：500ul plasmid solutions are taken to put from the 2nd pipe
Enter the 2nd pipe, adds no enzyme water 500ul, mixing；It goes 500ul plasmid solutions to be put into third pipe again from the 2nd pipe, adds no enzyme water
500ul, mixing；And so on.Finally often masterplates of the 1ul as quantitative fluorescent PCR is taken in pipe.
Q-PCR results are as follows：
The canonical plotting of drafting is shown in Fig. 2.
2.3 RNA reverse transcription PCRs react
Concentration mensuration is carried out to the total serum IgE of extraction, then quantifies RNA, is opened from 3 ' ends according to the reverse transcription process of setting
Begin to synthesize, obtains cDNA.
1. following reagent is added to successively in sterile no enzyme pipe：
2. mild mixing and brief centrifugation, 65 DEG C of incubation 5min；
3. following reagent is added in the above mixed liquor, final volume 20ul；
4. soft mixing and brief centrifugation are operated by following procedure：
Reaction completes gained cDNA (Complementary DNA) and is stored in -20 DEG C.
2.4 real-time fluorescence quantitative PCR
SYBR methods real-time fluorescence quantitative PCR (Quantitative Real-time PCR, q-PCR) reaction system：
iTaqTMUniversalGreen Supermix：
SYBR method real-time fluorescence quantitative PCR reaction steps：
The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction
After CT values (threshold cycle values), reference gene (pGL3) markization, meter is examined using unpaired t-test
Calculate P values.
Taqman probe method real-time fluorescence quantitative PCR reaction systems：
iTaqTMUniversal Probes Supermix：
Taqman probe real-time fluorescence quantitative PCRs (Quantitative Real-time PCR, q-PCR) reactant
System：
Taqman probe real-time fluorescence quantitative PCR reaction steps：
Confirm the expression intensity of each gene according to CT values (threshold cycle values), outer ginseng base after reaction
Because after (pGL3) markization, being examined using unpaired t-test and calculating P values.
2.5 statistical analysis
Statistical analysis is carried out using SPSS 13.0 and 5.0 softwares of Graphpad.
3 results
3.1 LOC284454, circMAN1A2 and circRNF13 high expression in patients with nasopharyngeal carcinoma
Experiment uses 100 patients with nasopharyngeal carcinoma and 51 normal control's serum, and experimental result is shown：With it is normal
Control group is compared, LOC284454, and circMAN1A2 and the circRNF13 up-regulated expression in patients with nasopharyngeal carcinoma are shown in Fig. 3.
To ensure experimental data authenticity and reliability, the error in experimentation is reduced, we then devise
The Taqman probes of LOC284454, circMAN1A2 and circRNF13, take the experimental method of Taqman probe q-PCR
Detect expression in patients with nasopharyngeal carcinoma of LOC284454, circMAN1A2 and circRNF13, and by normal control
Increase 121, such as Fig. 4.As a result it shows：Compared with Normal group serum, LOC284454, circMAN1A2 and
Expressions of the circRNF13 in patients with nasopharyngeal carcinoma is significantly raised, has statistical significance (P ＜ 0.001).
3.2 nasopharyngeal carcinoma ROC curves assess clinical value
The present invention verifies LOC284454, circMAN1A2 and circRNF13 and is raised in nasopharyngeal carcinoma, using ROC
The clinical value of curve assessment LOC284454, circMAN1A2 and circRNF13.
When AUC value is between 0.5 and 1.0, AUC>When 0.5, AUC shows that specificity is better with sensitivity closer to 1,
Clinical value is bigger.
It is bent to carry out ROC according to expression in nasopharyngeal carcinoma of LOC284454, circMAN1A2 and circRNF13 for we
Line analysis, such as Fig. 5.The AUC numerical value that ROC curve analyzes LOC284454 is 0.931；The AUC numerical value of circMAN1A2 is
0.911；The AUC numerical value of circRNF13 is 0.877；The AUC numerical value of the ROC curve analysis of three kinds of molecules of joint is 0.968, is said
Accuracy higher of the three kinds of molecules of bright joint on diagnosing tumor.
The single molecule ROC curve analyses of LOC284454
Area under curve (AUC)
The parameter analyzed:LOC284454
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The single molecule ROC curve analyses of CircMAN1A2
Area under curve (AUC)
The parameter analyzed:circMAN1A2
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The single molecule ROC curve analyses of circRNF13
Area under curve (AUC)
The parameter analyzed:circRNF13
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The tri- molecule ROC curve analyses of joint LOC284454, circRNF13 and circMAN1A2
Area under curve (AUC)
The parameter analyzed:LOC284454, circRNF13 and circMAN1A2
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
3.3 LOC284454, circMAN1A2 and circRNF13 high expression in oral cancer patient's serum
Experiment uses 55 oral cancer patient's serum and 121 normal control's serum, devises LOC284454,
The Taqman probes of circMAN1A2 and circRNF13 take the experimental method of Taqman probe q-PCR to detect
The expression of LOC284454, circMAN1A2 and circRNF13 in oral cancer patient's serum, as shown in Figure 6.As a result it shows
Show：Compared with Normal group serum, LOC284454, circMAN1A2 and circRNF13 are in oral cancer patient's serum
Expression is significantly raised, has statistical significance (P ＜ 0.001).
3.4 carcinoma of mouth ROC curves assess clinical value
The present invention verifies LOC284454, circMAN1A2 and the circRNF13 up-regulated expression in carcinoma of mouth, using ROC
The clinical value of curve assessment LOC284454, circMAN1A2 and circRNF13.
It is bent to carry out ROC according to expression in carcinoma of mouth of LOC284454, circMAN1A2 and circRNF13 for we
Line analysis, such as Fig. 7.The AUC numerical value that ROC curve analyzes LOC284454 is 0.698；The AUC numerical value of circMAN1A2 is
0.779, circRNF13 AUC numerical value is 0.731；The AUC numerical value of the ROC curve analysis of three kinds of molecules of joint is 0.863, is said
Accuracy higher of the three kinds of molecules of bright joint on diagnosing tumor.
The single molecule ROC curve analyses of IncLOC284454
Area under curve (AUC)
The parameter analyzed:LOC284454
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The single molecule ROC curve analyses of circMAN1A2
Area under curve (AUC)
The parameter analyzed:circMAN1A2
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The single molecule ROC curve analyses of circRNF13
Area under curve (AUC)
The parameter analyzed:circRNF13
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The tri- molecule ROC curve analyses of joint LOC284454, circRNF13 and circMAN1A2
Area under curve (AUC)
The parameter analyzed:LOC284454, circRNF13 and circMAN1A2
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
3.5 LOC284454, circMAN1A2 and circRNF13 high expression in thyroid cancer patients serum
We take Taqman probe q- in 57 thyroid cancer patients serum and 121 normal control's serum
The experimental method of PCR detects LOC284454, circMAN1A2 and the circRNF13 expression in thyroid cancer patients serum,
As a result it shows：Compared with Normal group, LOC284454, circMAN1A2 and circRNF13 are in thyroid cancer patients serum
Middle expression is significantly raised, has statistical significance (P ＜ 0.001), such as Fig. 8.
3.6 thyroid cancer ROC curves assess clinical value
The present invention verifies LOC284454, circMAN1A2 and the circRNF13 up-regulated expression in thyroid cancer, uses
ROC curve assesses the clinical value of LOC284454, circMAN1A2 and circRNF13.
We carry out ROC according to the expression of LOC284454, circMAN1A2 and circRNF13 in thyroid cancer
Tracing analysis, such as Fig. 9.The AUC numerical value that ROC curve analyzes LOC284454 is 0.834；The AUC numerical value of circMAN1A2 is
0.734, circRNF13 AUC numerical value is 0.798；The AUC numerical value of the ROC curve analysis of three kinds of molecules of joint is 0.881, is said
Accuracy higher of the three kinds of molecules of bright joint on diagnosing tumor.
The single molecule ROC curve analyses of LOC284454
Area under curve (AUC)
The parameter analyzed:LOC284454
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The single molecule ROC curve analyses of circMAN1A2
Area under curve (AUC)
The parameter analyzed:circMAN1A2
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The single molecule ROC curve analyses of circRNF13
Area under curve (AUC)
The parameter analyzed:circRNF13
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5
The tri- molecule ROC curve analyses of joint LOC284454, circRNF13 and circMAN1A2
Area under curve (AUC)
The parameter analyzed:LOC284454, circRNF13 and circMAN1A2
A. nonparametric is based on to assume
B. null hypothesis:True area=0.5.
Sequence table
<110>Central South University
<120>The application of three kinds of non-coding RNA reagents of detection 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
atcaggagag gag 553
<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
gccttctcat gatcagctcg 20
<210> 6
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 6
gtccaggata gacatagagc 20
<210> 7
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 7
gtgtagactt gtgtggctga 20
<210> 8
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 8
tccatcttgc tccaacaccc 20
<210> 9
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 9
tcgtctttcc gtgctccaaa 20
<210> 10
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 10
attacaggtg gctcaggtgt 20
<210> 11
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 11
cttcagtgtg cctcctcagt 20
<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 (10)
1. detecting the application of three kinds of non-coding RNA reagents, which is characterized in that the reagent is used to prepare tumour auxiliary diagnosis system
Agent, three kinds of non-coding RNAs are circular rna circMAN1A2, and sequence is as shown in SEQ No.1, circular rna circRNF13,
Its sequence is as shown in SEQ No.2, and long-chain non-coding RNA LOC284454, sequence is as shown in SEQ No.3.
2. application according to claim 1, which is characterized in that three kinds of non-coding RNA reagents of detection are three in detection serum
The reagent of kind non-coding RNA.
3. application according to claim 1 or 2, which is characterized in that tumor type includes：Nasopharyngeal carcinoma, carcinoma of mouth, thyroid gland
At least one of cancer.
4. application according to claim 1 or 2, which is characterized in that three kinds of non-coding RNA reagents of detection include real-time fluorescence
Quantitative detecting reagent.
5. application according to claim 4, which is characterized in that include to be used in the real time fluorescent quantitative detection reagent
Real time fluorescent quantitative detects the primer of circular rna circMAN1A2 expression：
Sense primer：5’-AGATGGGCAAAGATGGATTGA-3’
Downstream primer：5’-GCCTTCTCATGATCAGCTCG-3’；
And the primer for real time fluorescent quantitative detection circular rna circRNF13 expression：
Sense primer：5’-GTCCAGGATAGACATAGAGC-3’
Downstream primer：5’-GTGTAGACTTGTGTGGCTGA-3’；
And the primer for real time fluorescent quantitative detection long-chain non-coding RNA LOC284454 expression：
Sense primer：5’-ATTACAGGTGGCTCAGGTGT-3’
Downstream primer：5’-CTTCAGTGTGCCTCCTCAGT-3’.
6. a kind of kit for tumour auxiliary diagnosis, which is characterized in that containing circular rna in serum can be detected
The reagent of circMAN1A2, circular rna circRNF13 and long-chain non-coding RNA LOC284454；The circular rna
The sequence of circMAN1A2 is as shown in SEQ No.1, and the sequence of the circular rna circRNF13 is as shown in SEQ No.2, institute
The sequence of the long-chain non-coding RNA LOC284454 stated is as shown in SEQ No.3.
7. kit according to claim 6, which is characterized in that described can detect circular rna in serum
Ring can be expanded by containing in the reagent of circMAN1A2, circular rna circRNF13 and long-chain non-coding RNA LOC284454
The primer of shape RNA circMAN1A2, circular rna circRNF13 and long-chain non-coding RNA LOC284454.
8. kit according to claim 7, which is characterized in that
The primer of circular rna circMAN1A2 can be expanded：
Sense primer：5’-AGATGGGCAAAGATGGATTGA-3’
Downstream primer：5’-GCCTTCTCATGATCAGCTCG-3’；
The primer of circular rna circRNF13 can be expanded：
Sense primer：5’-GTCCAGGATAGACATAGAGC-3’
Downstream primer：5’-GTGTAGACTTGTGTGGCTGA-3’；
The primer of long-chain non-coding RNA LOC284454 can be expanded：
Sense primer：5’-ATTACAGGTGGCTCAGGTGT-3’
Downstream primer：5’-CTTCAGTGTGCCTCCTCAGT-3’.
9. kit according to claim 7, which is characterized in that the kit further includes：Internal reference pGL3 primers：
Sense primer：5’-TCCATCTTGCTCCAACACCC-3’
Downstream primer：5’-TCGTCTTTCCGTGCTCCAAA-3’.
10. according to the kit described in claim 7 or 8 or 9, which is characterized in that the kit further includes：
The mating Taqman probes of the primer of circular rna circMAN1A2 expression are detected for real time fluorescent quantitative：5’-ROX-
CAAAGATGGATTGAAGACAACCTTGATTTCAGTGTG-BHQ2-3’；
The mating Taqman probes of the primer of circular rna circRNF13 expression are detected for real time fluorescent quantitative：5’-Cy5-
AGGATAGACATAGAGCTAGAAGAAACAGACTTCGT-BHQ2-3’；Long-chain non-coding RNA is detected for real time fluorescent quantitative
The mating Taqman probes of primer of LOC284454 expression：5’-FAM–CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1-
3’
The mating Taqman probes of internal reference pGL3 primers：5’-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1-3’.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112870364A (en) * | 2021-04-12 | 2021-06-01 | 中南大学湘雅三医院 | Application of circ-RNF13-TRIM41-p53 complex as target site detection preparation |
| CN113101369A (en) * | 2021-04-12 | 2021-07-13 | 中南大学湘雅三医院 | Application of circular RNA circ-RNF13 as target site inhibitor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441581A (en) * | 2016-01-29 | 2016-03-30 | 中南大学 | Application of reagent for detecting long-chain non-coding RNA LOC284454 expression quantity in tongue cancer tissue |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441581A (en) * | 2016-01-29 | 2016-03-30 | 中南大学 | Application of reagent for detecting long-chain non-coding RNA LOC284454 expression quantity in tongue cancer tissue |
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| WANG ET AL.: "CircRNF13 regulates the invasion and metastasis in lung adenocarcinoma by targeting miR-93-5p", 《GENE》 * |
| ZHONG C.F.等: "Heterogeneous circRNA expression profiles and regulatory functions among HEK293T single cells", 《SCIENTIFIC REPORTS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112870364A (en) * | 2021-04-12 | 2021-06-01 | 中南大学湘雅三医院 | Application of circ-RNF13-TRIM41-p53 complex as target site detection preparation |
| CN113101369A (en) * | 2021-04-12 | 2021-07-13 | 中南大学湘雅三医院 | Application of circular RNA circ-RNF13 as target site inhibitor |
| CN113101369B (en) * | 2021-04-12 | 2022-03-11 | 中南大学湘雅三医院 | Application of circular RNA circ-RNF13 as target site inhibitor |
| CN112870364B (en) * | 2021-04-12 | 2022-04-12 | 中南大学湘雅三医院 | Application of circ-RNF13-TRIM41-p53 complex as target site detection preparation |
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