CN110643695B - Systemic Lupus Erythematosus (SLE) circular RNA marker and detection reagent - Google Patents
Systemic Lupus Erythematosus (SLE) circular RNA marker and detection reagent Download PDFInfo
- Publication number
- CN110643695B CN110643695B CN201810682501.0A CN201810682501A CN110643695B CN 110643695 B CN110643695 B CN 110643695B CN 201810682501 A CN201810682501 A CN 201810682501A CN 110643695 B CN110643695 B CN 110643695B
- Authority
- CN
- China
- Prior art keywords
- seq
- primer
- sequence shown
- reagent
- primer pair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- C12Q2600/118—Prognosis of disease development
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of systemic lupus erythematosus diagnosis research, and particularly provides a series of systemic lupus erythematosus circular RNA markers and detection reagents which can be used for blood sample detection.circUIMC1，circPPP1CB，circKIAA0368， circCAMSAP1，circPVT1，circTBCDThe expression level of the circular RNA in peripheral blood mononuclear cells of the systemic lupus erythematosus patient is obviously lower than that of a normal person, and the circular RNA can be used as a molecular marker of the systemic lupus erythematosus. The detection reagent of the series of molecular markers is a specific primer pair. The detection reagent can specifically detect that the expression level of corresponding circular RNA in peripheral blood mononuclear cells of a systemic lupus erythematosus patient is obviously lower than that of a normal person. The circular RNA stably exists in cells and is not easy to degrade, and the gene detection reagent has the advantages of specificity, sensitivity, rapidness, simplicity and convenience.
Description
Technical Field
The invention belongs to the field of systemic lupus erythematosus diagnosis research, and particularly relates to a systemic lupus erythematosus circular RNA marker and a detection reagent.
Background
Systemic Lupus Erythematosus (SLE) is a typical multi-organ, multi-system autoimmune disease, and its clinical and immunological phenotypes are very complex and diverse, including immune tolerance deficiency, lymphocyte function regulation and apoptosis disorder, complement deficiency and immune complex clearance disorder, cytokine secretion regulation disorder, etc., and almost cover the disorder of the whole immune system. Clinically, it is manifested as damage to various organs such as kidney, nervous system, mental system, and blood system. If early diagnosis can be performed before the SLE patient is affected by important organs, the method has great significance for preventing and treating SLE, improving the life quality of the patient and improving the productivity of the patient. However, most of the current markers for biological diagnosis are biochemical and immunological changes occurring after organ damage, and early diagnosis of organ involvement in SLE patients is impossible.
Currently, the etiology and pathogenesis of SLE are not completely clear, and are influenced by many aspects such as family inheritance, sex hormone disorder, environmental factors and the like. Therefore, specific treatment means is still lacked, and the prevention and treatment level of the disease cannot be fundamentally improved.
Circular RNAs (circRNAs) are a recently discovered class of non-coding RNA molecules. Unlike conventional linear RNA, circular RNA does not have a 5 'terminal cap and a 3' terminal poly (A) tail, and forms a closed circular structure with covalent bonds. Recent studies indicate that circular RNA is mainly generated through reverse-splicing (back-splicing), widely exists in various biological cells, has extremely high structural stability, is difficult to be degraded by exonuclease (exonuclease), and has the characteristics of tissue and space-time specificity in expression. These characteristics make circRNA have wide prospects in the development and application of disease diagnosis and treatment methods.
Currently, there is no clinically available diagnostic index for SLE with high sensitivity and specificity, and even though autoantibodies such as anti-ANA antibodies have diagnostic sensitivity for SLE of 95%, their diagnostic specificity is relatively low, only 65%. For this reason, the patient must examine many laboratory indexes in combination to assist in diagnosing SLE, which results in a significant increase in the diagnosis cost of the disease and burdens the patient. Therefore, the development of a new SLE diagnostic marker is necessary, and the SLE diagnostic marker has important significance for improving the diagnosis and treatment level of SLE.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a systemic lupus erythematosus circular RNA marker and a detection reagent.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
in a first aspect of the invention, there is provided the use of any one or more of circUIMC1, circPPP1CB, circKIAA0368, circcamap 1, circPVT1 and circTBCD in combination for the preparation or screening of a test agent for the detection of systemic lupus erythematosus. By plurality is meant two, three, four, five or six.
In one embodiment, any one or more of circUIMC1, circPPP1CB, circKIAA0368, circcamap 1, circPVT1, and circTBCD are combined as biomarkers. By plurality is meant two, three, four, five or six.
In one embodiment, the systemic lupus erythematosus detection reagent is used for the judgment and diagnosis of systemic lupus erythematosus.
It should be noted that the systemic lupus erythematosus detection reagent is not limited to be necessarily in a liquid form.
In one embodiment, the systemic lupus erythematosus detection reagent comprises any one or more of: (1) a reagent specifically recognizing circUIMC1, (2) a reagent specifically recognizing circPPP1CB, (3) a reagent specifically recognizing circKIAA0368, (4) a reagent specifically recognizing circcamasp 1, (5) a reagent specifically recognizing circPVT1, and (6) a reagent specifically recognizing circTBCD. The terms refer to two, three, four, five or six terms.
circUIMC1 circID in the circcpedia v2 database is HSA _ circcpedia _51248.
circPPP1CB has a circID in the circcpedia v2 database as HSA _ circcpedia _40657.
circID in circKIAA0368 database in circcpedia v2 is HSA _ circcpedia _342200.
circID of circcamap 1 in the circcpedia v2 database is HSA _ circcpedia _63391.
The circID of circPVT1 in the circcpedia v2 database is HSA _ circcpedia _60029.
circID in circTBCD in the circcpedia v2 database is HSA _ circcpedia _341917.
In one embodiment, the reagent that specifically recognizes circUIMC1 is selected from a primer pair that specifically amplifies circUIMC 1. For example, the primer pair for specifically amplifying circUIMC1 comprises an upstream primer with a sequence shown as SEQ ID No.1 and a downstream primer with a sequence shown as SEQ ID No. 2.
In one embodiment, the reagent that specifically recognizes circPPP1CB is selected from a primer pair that specifically amplifies circPPP1 CB. For example, the primer pair for specifically amplifying circPPP1CB comprises an upstream primer with a sequence shown in SEQ ID NO.3 and a downstream primer with a sequence shown in SEQ ID NO. 4.
In one embodiment, the reagent specifically recognizing circKIAA0368 is selected from a primer pair specifically amplifying circKIAA 0368. For example, the primer pair for specifically amplifying circKIAA0368 comprises an upstream primer with a sequence shown as SEQ ID NO.5 and a downstream primer with a sequence shown as SEQ ID NO. 6.
In one embodiment, the reagent that specifically recognizes circcamap 1 is selected from a primer pair that specifically amplifies circcamap 1. For example, the primer pair for specifically amplifying circcamap 1 comprises an upstream primer with a sequence shown as SEQ ID NO. 7and a downstream primer with a sequence shown as SEQ ID NO. 8.
In one embodiment, the reagent specifically recognizing circPVT1 is selected from a primer pair specifically amplifying circPVT 1. For example, the primer pair for specifically amplifying circPVT11 comprises an upstream primer with a sequence shown as SEQ ID NO. 9and a downstream primer with a sequence shown as SEQ ID NO. 10.
In one embodiment, the reagent that specifically recognizes circTBCD is selected from a primer pair that specifically amplifies circTBCD. For example, the primer pair for specifically amplifying circTBCD comprises an upstream primer with a sequence shown as SEQ ID NO.11 and a downstream primer with a sequence shown as SEQ ID NO. 12.
In the second aspect of the invention, any one or more of a reagent specifically recognizing circUIMC1, a reagent specifically recognizing circPPP1CB, a reagent specifically recognizing circKIAA0368, a reagent specifically recognizing circcamasp 1, a reagent specifically recognizing circPVT1 and a reagent specifically recognizing circTBCD is combined for preparing a systemic lupus erythematosus detection kit. The terms refer to two, three, four, five or six terms.
In one embodiment, any one or more of circUIMC1, circPPP1CB, circKIAA0368, circcamap 1, circPVT1 and circTBCD are combined as a biomarker. By plurality is meant two, three, four, five or six.
In one embodiment, the kit is used for the determination and diagnosis of systemic lupus erythematosus.
The reagent specifically recognizing circUIMC1, the reagent specifically recognizing circPPP1CB, the reagent specifically recognizing circKIAA0368, the reagent specifically recognizing circcamap 1, the reagent specifically recognizing circPVT1, and the reagent specifically recognizing circTBCD are not limited to liquid forms.
In one embodiment, the reagent that specifically recognizes circUIMC1 is selected from a primer pair that specifically amplifies circUIMC 1. For example, the primer pair for specifically amplifying circUIMC1 comprises an upstream primer with a sequence shown as SEQ ID No.1 and a downstream primer with a sequence shown as SEQ ID No. 2.
In one embodiment, the reagent that specifically recognizes circPPP1CB is selected from a primer pair that specifically amplifies circPPP1 CB. For example, the primer pair for specifically amplifying circPPP1CB comprises an upstream primer with a sequence shown in SEQ ID NO.3 and a downstream primer with a sequence shown in SEQ ID NO. 4.
In one embodiment, the reagent specifically recognizing circKIAA0368 is selected from a primer pair specifically amplifying circKIAA 0368. For example, the primer pair for specifically amplifying circKIAA0368 comprises an upstream primer with a sequence shown as SEQ ID NO.5 and a downstream primer with a sequence shown as SEQ ID NO. 6.
In one embodiment, the reagent that specifically recognizes circcamap 1 is selected from a primer pair that specifically amplifies circcamap 1. For example, the primer pair for specifically amplifying circcamasp 1 comprises an upstream primer with a sequence shown as SEQ ID NO. 7and a downstream primer with a sequence shown as SEQ ID NO. 8.
In one embodiment, the reagent specifically recognizing circPVT1 is selected from a primer pair specifically amplifying circPVT 1. For example, the primer pair for specifically amplifying circPVT11 comprises an upstream primer with a sequence shown as SEQ ID NO. 9and a downstream primer with a sequence shown as SEQ ID NO. 10.
In one embodiment, the reagent that specifically recognizes circTBCD is selected from a primer pair that specifically amplifies circTBCD. For example, the primer pair for specifically amplifying circTBCD comprises an upstream primer with a sequence shown as SEQ ID NO.11 and a downstream primer with a sequence shown as SEQ ID NO. 12.
In a third aspect of the invention, a systemic lupus erythematosus assay kit is provided, wherein the kit comprises any one or more of the following: (1) a reagent specifically recognizing circUIMC1, (2) a reagent specifically recognizing circPPP1CB, (3) a reagent specifically recognizing circKIAA0368, (4) a reagent specifically recognizing circcamasp 1, (5) a reagent specifically recognizing circPVT1, and (6) a reagent specifically recognizing circTBCD. The terms refer to two, three, four, five or six terms.
In one embodiment, the kit is used for judging and diagnosing systemic lupus erythematosus.
In one embodiment, any one or more of circUIMC1, circPPP1CB, circKIAA0368, circcamap 1, circPVT1 and circTBCD are combined as a biomarker. By plurality is meant two, three, four, five or six.
The agent specifically recognizing circUIMC1, the agent specifically recognizing circPPP1CB, the agent specifically recognizing circKIAA0368, the agent specifically recognizing circcamap 1, the agent specifically recognizing circPVT1, and the agent specifically recognizing circTBCD are not limited to liquid forms.
In one embodiment, the agent that specifically recognizes circUIMC1 is selected from a primer pair that specifically amplifies circUIMC 1. For example, the primer pair for specifically amplifying circUIMC1 comprises an upstream primer with a sequence shown as SEQ ID No.1 and a downstream primer with a sequence shown as SEQ ID No. 2.
In one embodiment, the reagent that specifically recognizes circPPP1CB is selected from a primer pair that specifically amplifies circPPP1 CB. For example, the primer pair for specifically amplifying circPPP1CB comprises an upstream primer with a sequence shown in SEQ ID NO.3 and a downstream primer with a sequence shown in SEQ ID NO. 4.
In one embodiment, the reagent specifically recognizing circKIAA0368 is selected from a primer pair specifically amplifying circKIAA 0368. For example, the primer pair for specifically amplifying circKIAA0368 comprises an upstream primer with a sequence shown as SEQ ID NO.5 and a downstream primer with a sequence shown as SEQ ID NO. 6.
In one embodiment, the reagent that specifically recognizes circcamap 1 is selected from a primer pair that specifically amplifies circcamap 1. For example, the primer pair for specifically amplifying circcamasp 1 comprises an upstream primer with a sequence shown as SEQ ID NO. 7and a downstream primer with a sequence shown as SEQ ID NO. 8.
In one embodiment, the reagent specifically recognizing circPVT1 is selected from a primer pair specifically amplifying circPVT 1. For example, the primer pair for specifically amplifying circPVT11 comprises an upstream primer with a sequence shown as SEQ ID NO. 9and a downstream primer with a sequence shown as SEQ ID NO. 10.
In one embodiment, the reagent that specifically recognizes circTBCD is selected from a primer pair that specifically amplifies circTBCD. For example, the primer pair for specifically amplifying circTBCD comprises an upstream primer with a sequence shown as SEQ ID NO.11 and a downstream primer with a sequence shown as SEQ ID NO. 12.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a series of systemic lupus erythematosus circular RNA markers and detection reagents which can be used for blood sample detection. The series of circular RNA molecular markers are as follows: circUIMC1, circPPP1CB, circKIAA0368, circCAMSAP1, circPVT1, circTBCD. The expression level of the circular RNA in peripheral blood mononuclear cells of the systemic lupus erythematosus patient is obviously lower than that of a normal person, and the circular RNA can be used as a molecular marker of the systemic lupus erythematosus. The detection reagent of the series of molecular markers is a specific primer pair. The detection reagent can specifically detect that the expression level of corresponding circular RNA in peripheral blood mononuclear cells of a systemic lupus erythematosus patient is obviously lower than that of a normal person. The gene detection reagent has the advantages of specificity, sensitivity, rapidness, simplicity and convenience.
Drawings
FIG. 1: is a position diagram of gene sequences of 6 circular RNA molecular markers and a PCR primer recognition sequence.
FIG. 2 is a schematic diagram: 6 circular RNA molecular markers are combined to be used as a biomarker and a PCR product detection result.
FIG. 3: the results of detecting the expression level of the circular RNA corresponding to the peripheral blood mononuclear cell sample of the systemic lupus erythematosus patient by using the specific PCR primers in the table 1 are shown.
FIG. 4: the receiver operating characteristic curve (ROC curve) was obtained using 6 circular RNAs, respectively.
Detailed Description
According to the invention, through research, the expression level of the gene UTX and the gene EFNB1 is found to have correlation with the prognosis and survival time of the human systemic lupus erythematosus for the first time, and the prognosis of the systemic lupus erythematosus patient with low expression of the gene UTX and/or high expression of the gene EFNB1 is worse.
Based on the above-mentioned novel findings of the present inventors, the UTX gene and/or the gene EFNB1 gene can be used as a biomarker of systemic lupus erythematosus. The nucleic acid or protein detection kit for detecting the gene UTX andor the gene EFNB1 can be used for detecting the expression level of the gene UTX and the gene EFNB1 in the systemic lupus erythematosus, and the results can be used for evaluating the malignancy degree of the human systemic lupus erythematosus, predicting the possibility of CNS invasion of the systemic lupus erythematosus and realizing the molecular typing and early diagnosis of the malignant systemic lupus erythematosus.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, the invention may be practiced using any method, device, and material that is similar or equivalent to the methods, devices, and materials described in examples herein, in addition to those described in prior art practice and the description herein.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature and are described in particular in Sambrook et al, molecular CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and Third edition,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHOD IN ENZYMOLOGY, vol.304, chromatin (P.M. Wassarman and A.P. Wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.
Example 1 Gene sequence of circular RNA molecular marker and PCR primer recognition sequence position diagram
A circular RNA molecular marker combination of systemic lupus erythematosus, comprising 6 circular RNA molecular markers, circUIMC1, circPPP1CB, circKIAA0368, circCAMSAP1, circPVT1, circTBCD, the on-chromosome location (FIG. 1) and the sequence thereof are obtained in the CIRCexplorer database (http:// yanglab. Githu. Io/CIRCCexplor /). The detection reagent of the systemic lupus erythematosus circular RNA molecular marker combination is a plurality of pairs of specific primers, the plurality of pairs of primers are designed through research and analysis of the circular RNA cyclization sites, and the primers are synthesized by Shanghai Boshang biological Limited company. The nucleotide sequence of the specific primers is as follows:
TABLE 1
Example 2 detection results of PCR products with 6 circular RNA molecular markers as combined biomarkers
Step 1: extraction of PA-1 cellular RNA
The culture was first decanted, the cells washed twice with PBS and the liquid blotted dry. Cells were placed on ice and Trizol reagent was added (10 cm dish plus 1ml 6cm dish plus 0.5ml 6 well plate plus 0.2ml per void). The liquid became viscous, the cells detached from the wall, and fully blown to be clear. The cell lysate was aspirated into DEPC-treated EP tube, 0.2 volume of chloroform was added, shaken for 15 seconds, inverted several times, and allowed to stand at room temperature for 2-3 minutes. Centrifugation at 12000g for 15 minutes at 4 ℃ revealed liquid stratification: bottom-red phenol chloroform phase; middle layer-pink interphase; upper layer-colorless liquid phase, with RNA all in this phase. The supernatant was carefully transferred to a new EP tube, the middle layer was aspirated, an equal volume of isopropanol was added, inverted several times, and allowed to stand at room temperature for 10 minutes. Centrifuging at 12000g for 15 min at 4 ℃, discarding the supernatant, and obtaining a milky small precipitate which is the RNA at the bottom of the EP tube. DEPC-ethanol wash 2 times, adding 1ml75% ethanol. The supernatant was blotted dry and air dried for 10 minutes. Adding 20-30 μ l DEPC water, mixing, and dissolving RNA. And (3) measuring the concentration: a260/280 is between 1.8 and 2.0, and A260/230 is about 2.0; a260 Between 0.1 and 1, the samples were stored at-80 ℃.
And 2, step: reverse transcription to obtain cDNA
(1) DNase treatment of RNA, the reaction system is as follows:
DEPC-treated water
RQ1 DNase10×Reaction Buffer 1μl
RQ1 RNase-FreeDNase 1U/μgRNA
the reaction was carried out at 37 ℃ for 30 minutes, and 1. Mu.l of stop Buffer was added thereto, followed by reaction at 65 ℃ for 10 minutes.
(2) To each reaction, 0.5. Mu.g of random primer was added, reacted at 72 ℃ for 5 minutes, and allowed to stand on ice for 2 minutes.
(3) The reverse transcription reaction system is as follows:
the reaction was carried out at 37 ℃ for 60 minutes and at 72 ℃ for 10 minutes.
And step 3: amplification of DNA fragments of interest
And amplifying the DNA fragment to be detected by using the specific PCR primers in the table 1. The PCR reaction system is as follows:
the PCR reaction conditions were as follows:
Stage 1：95℃，5min
Stage 2：95℃，30s
55℃，30s 34 cycles
72℃，30s
Stage 3：72℃，5min
and 4, step 4: DNA electrophoresis detection
The DNA was mixed in 1 XDNA loading buffer, the electrophoresis solution was 1 XTAE solution, and 2. Mu.g/ml EB was contained in the Agrose gel. The DNA electrophoresis voltage is generally 5-8V/cm. The electrophoresis time is determined according to the size of the DNA fragment. The sizes of the resolvable DNA fragments were different in gels of different concentrations. 1% of Agrose gel is selected for the experiment and is electrophoresed for 20 minutes. The electrophoresis results are shown in FIG. 2.
Example 3 detection results of circular RNA expression levels in peripheral blood mononuclear cell samples of systemic lupus erythematosus patients using specific PCR primers in Table 1
Step 1: obtaining peripheral blood mononuclear cells of normal people and systemic lupus erythematosus patients
We obtained peripheral blood samples from Renji Hospital, shanghai, of 8 normal persons and 8 patients with systemic lupus erythematosus. And separating the peripheral blood mononuclear cells by a density gradient centrifugation method for subsequent detection.
Step 2: RNA extraction and reverse transcription
Peripheral blood mononuclear cell RNA from normal and lupus erythematosus patients was extracted and reverse transcribed into cDNA as described in example 2 above.
And step 3: fluorescent quantitative Q-PCR
Total RNA was extracted as in example 2 above. Reverse transcription was performed as described in example 2 above using random primers. Real-time quantitative PCR reactions were performed in a Biorad realtime PCR instrument. The reaction system is as follows:
the reaction conditions were as follows:
data is according to 2 -ΔΔCT Methods were performed (Kenneth j. Livak, thomas d. Schmittgen. Method,25, 2001.
As can be seen from FIG. 3, the expression level of these 6 circular RNAs in peripheral blood mononuclear cells of SLE patients is significantly lower than that of normal persons. Significant differences were demonstrated using independent sample T-test (T-test) in which the P-value for 5 circular RNAs was less than 0.05. As shown in FIG. 4, the 6 circular RNAs were used to prepare the receiver operating characteristic curves (ROC curves), and the area under the curves (AUC) were all above 0.9, indicating that the 6 circular RNAs as markers have very high diagnostic sensitivity and specificity for the identification of SLE by the peripheral blood mononuclear cell sample.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Sequence listing
<110> Shanghai Life science research institute of Chinese academy of sciences
Affiliated ren Ji Hospital of Shanghai university medical college
<120> systemic lupus erythematosus circular RNA marker and detection reagent
<130> 183906
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tccacccaca aagattgaac g 21
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aacactttgt gaagcaggta c 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttcctttact tttggagctg a 21
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cagccttgca catccttatc t 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aagtatttgg aatgcgttgg t 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tatacaccag atctggctgg c 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tacactgtgg agatgatcag c 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggccacctgg tcccctttca g 21
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gagggttgag atctctgttt a 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gaagatcact gtaaatccat c 21
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gtgcagggcc tgaagcagat t 21
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ctgaactcgg gtgcctgctg g 21
Claims (14)
1. Specific recognitioncircUIMC1Reagent (2) and specific recognitioncircPPP1CBReagent (2) and specific recognitioncircKIAA0368Reagent (2) and specific recognitioncircCAMSAP1Reagent (2) and specific recognitioncircPVT1And specific recognitioncircTBCDThe use of any one or more of the agents of (a) in combination for the preparation or screening of a test agent for systemic lupus erythematosus detection;
the above-mentionedcircUIMC1circID in the circcpedia v2 database is HSA _ circcpedia _51248;
the above-mentionedcircPPP1CBcircID in the circcpedia v2 database is HSA _ circcpedia _40657;
the describedcircKIAA0368circID in the circcpidia v2 database is HSA _ circcpidia _342200;
the above-mentionedcircCAMSAP1circID in the circcpedia v2 database is HSA _ circcpedia _63391;
the above-mentionedcircPVT1circID in the circcpidia v2 database is HSA _ circcpidia _60029;
the above-mentionedcircTBCDThe circID in the circcpedia v2 database is HSA _ circedia _341917.
2. Use according to claim 1,circUIMC1、circPPP1CB、circKIAA0368、 circCAMSAP1、circPVT1andcircTBCDas a biomarker.
3. The use of claim 1, wherein the systemic lupus erythematosus detection reagent is used for the judgment and diagnosis of systemic lupus erythematosus.
4. Use according to claim 1, further comprising any one or more of the following features: (1) The specific recognitioncircUIMC1The reagent(s) is selected from the group consisting of specific amplificationcircUIMC1The primer pair of (1); (2) The specific recognitioncircPPP1CBThe reagent(s) is selected from the group consisting of specific amplificationcircPPP1CBThe primer pair of (1); (3) The specific recognitioncircKIAA0368The reagent(s) is selected from the group consisting of specific amplificationcircKIAA0368The primer pair of (1); (4) The specific recognitioncircCAMSAP1The reagent is selected from specific amplificationcircCAMSAP1The primer pair of (4); (5) The specific recognitioncircPVT1The reagent is selected from specific amplificationcircPVT1The primer pair of (1); (6) The specific recognitioncircTBCDThe reagent(s) is selected from the group consisting of specific amplificationcircTBCDThe primer set (4).
5. Use according to claim 1, further comprising any one or more of the following features: (1) Specific amplificationcircUIMC1The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.1 and a downstream primer with a sequence shown as SEQ ID NO. 2; (2) Specific amplificationcircPPP1CBThe primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.3 and a downstream primer with a sequence shown as SEQ ID NO. 4; (3) Specific amplificationcircKIAA0368The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.5 and a downstream primer with a sequence shown as SEQ ID NO. 6; (4) Specific amplificationcircCAMSAP1The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO. 7and a downstream primer with a sequence shown as SEQ ID NO. 8; (5) Specific amplificationcircPVT11The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO. 9and a downstream primer with a sequence shown as SEQ ID NO. 10; (6) Specific amplificationcircTBCDThe primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.11 and a downstream primer with a sequence shown as SEQ ID NO. 12.
6. Specific recognitioncircUIMC1Of (2)、Specific recognitioncircPPP1CBOf (2)、Specific recognitioncircKIAA0368Of (2)、Specific recognitioncircCAMSAP1Of (2)、Specific recognitioncircPVT1And specific recognitioncircTBCDAny one or more of the reagents are jointly used for preparing a systemic lupus erythematosus detection kit;
the above-mentionedcircUIMC1circID in the circcpedia v2 database is HSA _ circcpedia _51248;
the above-mentionedcircPPP1CBcircID in the circcpedia v2 database is HSA _ circcpedia _40657;
the above-mentionedcircKIAA0368circID in the circcpidia v2 database is HSA _ circcpidia _342200;
the above-mentionedcircCAMSAP1circID in CIRCpedia v2 database is HSA _ CIRCpedia _63391;
the above-mentionedcircPVT1circID in the circID v2 database is HSA _ circID _60029;
the above-mentionedcircTBCDThe circID in the circcpedia v2 database is HSA _ circedia _341917.
7. Use according to claim 6,circUIMC1、circPPP1CB、circKIAA0368、 circCAMSAP1、circPVT1andcircTBCDas a biomarker.
8. The use of claim 6, wherein the systemic lupus erythematosus detection kit is used for judging and diagnosing systemic lupus erythematosus.
9. Use according to claim 6, further comprising any one or more of the following features: (1) The specific recognitioncircUIMC1The reagent is selected from specific amplificationcircUIMC1The primer pair of (1); (2) The specific recognitioncircPPP1CBThe reagent is selected from specific amplificationcircPPP1CBThe primer pair of (1); (3) The specificity isIdentificationcircKIAA0368The reagent is selected from specific amplificationcircKIAA0368The primer pair of (4); (4) The specific recognitioncircCAMSAP1The reagent is selected from specific amplificationcircCAMSAP1The primer pair of (1); (5) The specific recognitioncircPVT1The reagent is selected from specific amplificationcircPVT1The primer pair of (1); (6) The specific recognitioncircTBCDThe reagent is selected from specific amplificationcircTBCDThe primer set (2) of (1).
10. Use according to claim 9, further comprising any one or more of the following features: (1) Specific amplificationcircUIMC1The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.1 and a downstream primer with a sequence shown as SEQ ID NO. 2; (2) Specific amplificationcircPPP1CBThe primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.3 and a downstream primer with a sequence shown as SEQ ID NO. 4; (3) Specific amplificationcircKIAA0368The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.5 and a downstream primer with a sequence shown as SEQ ID NO. 6; (4) Specific amplificationcircCAMSAP1The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO. 7and a downstream primer with a sequence shown as SEQ ID NO. 8; (5) Specific amplificationcircPVT11The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO. 9and a downstream primer with a sequence shown as SEQ ID NO. 10; (6) Specific amplificationcircTBCDThe primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.11 and a downstream primer with a sequence shown as SEQ ID NO. 12.
11. A systemic lupus erythematosus detection kit, the kit comprising any one or more of the following: (1) Specific recognitioncircUIMC1Reagent (2) for specific recognitioncircPPP1CBReagent (3) for specific recognitioncircKIAA0368Reagent (4) for specific recognitioncircCAMSAP1And (5) specific recognitionTrials on circTBCD Agent for treating diabetes。
12. The systemic lupus erythematosus detection kit according to claim 11, wherein the systemic lupus erythematosus detection kit is used for judgment and diagnosis of systemic lupus erythematosus.
13. The systemic lupus erythematosus detection kit according to claim 11, further comprising any one or more of the following features: (1) The specific recognitioncircUIMC1The reagent(s) is selected from the group consisting of specific amplificationcircUIMC1The primer pair of (1); (2) The specific recognitioncircPPP1CBThe reagent is selected from specific amplificationcircPPP1CBThe primer pair of (4); (3) The specific recognitioncircKIAA0368The reagent is selected from specific amplificationcircKIAA0368The primer pair of (4); (4) The specific recognitioncircCAMSAP1The reagent(s) is selected from the group consisting of specific amplificationcircCAMSAP1The primer pair of (1); (5) The specific recognitioncircTBCDThe reagent is selected from specific amplificationcircTBCDThe primer set (4).
14. The systemic lupus erythematosus detection kit according to claim 13, further comprising any one or more of the following features: (1) Specific amplificationcircUIMC1The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.1 and a downstream primer with a sequence shown as SEQ ID NO. 2; (2) Specific amplificationcircPPP1CBThe primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.3 and a downstream primer with a sequence shown as SEQ ID NO. 4; (3) Specific amplificationcircKIAA0368The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.5 and a downstream primer with a sequence shown as SEQ ID NO. 6; (4) Specific amplificationcircCAMSAP1The primer pair comprises an upstream primer with a sequence shown as SEQ ID NO. 7and a downstream primer with a sequence shown as SEQ ID NO. 8; (5) Specific amplificationcircTBCDThe primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.11 and a downstream primer with a sequence shown as SEQ ID NO. 12.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810682501.0A CN110643695B (en) | 2018-06-27 | 2018-06-27 | Systemic Lupus Erythematosus (SLE) circular RNA marker and detection reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810682501.0A CN110643695B (en) | 2018-06-27 | 2018-06-27 | Systemic Lupus Erythematosus (SLE) circular RNA marker and detection reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110643695A CN110643695A (en) | 2020-01-03 |
| CN110643695B true CN110643695B (en) | 2023-02-17 |
Family
ID=68988901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810682501.0A Active CN110643695B (en) | 2018-06-27 | 2018-06-27 | Systemic Lupus Erythematosus (SLE) circular RNA marker and detection reagent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110643695B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363802A (en) * | 2020-03-31 | 2020-07-03 | 中国科学院昆明动物研究所 | CircRNA small molecule marker for indicating health aging key pathway and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101230386A (en) * | 2007-04-28 | 2008-07-30 | 廖凌虹 | Use of human heterogeneous substance metabolic enzymes gene mononucleotide polymorphism in diagnosing and treating systemic lupus erythematosus |
| CN107385013A (en) * | 2016-05-17 | 2017-11-24 | 戴勇 | The processing method of systemic loupus erythematosus circular rna |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7608395B2 (en) * | 2005-09-15 | 2009-10-27 | Baylor Research Institute | Systemic lupus erythematosus diagnostic assay |
| US20070238094A1 (en) * | 2005-12-09 | 2007-10-11 | Baylor Research Institute | Diagnosis, prognosis and monitoring of disease progression of systemic lupus erythematosus through blood leukocyte microarray analysis |
-
2018
- 2018-06-27 CN CN201810682501.0A patent/CN110643695B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101230386A (en) * | 2007-04-28 | 2008-07-30 | 廖凌虹 | Use of human heterogeneous substance metabolic enzymes gene mononucleotide polymorphism in diagnosing and treating systemic lupus erythematosus |
| CN107385013A (en) * | 2016-05-17 | 2017-11-24 | 戴勇 | The processing method of systemic loupus erythematosus circular rna |
Non-Patent Citations (5)
| Title |
|---|
| Circular RNA expression profile and potential function of hsa_circ_0045272 in systemic lupus erythematosus;Lian-Ju Li 等;《Immunology》;20180523;第155卷(第1期);第137-149页 * |
| Circular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer;JieChen 等;《Cancer Letters》;20171231;第388卷;图2、补充表S2 * |
| Comprehensive circular RNA profiles in plasma reveals that circular RNAs can be used as novel biomarkers for systemic lupus erythematosus;Haixia Li 等;《Clinica Chimica Acta》;20180220;第480卷;第17-25页 * |
| Structure and Degradation of Circular RNAs Regulate PKR Activation in Innate Immunity;Chu-Xiao Liu 等;《Cell》;20190502;第177卷;第865-880页 * |
| 环状RNA在系统性红斑狼疮患者中差异表达的研究;喻祥琪;《中国优秀博硕士学位论文全文数据库（硕士）医药卫生科技辑》;20170215(第2期);第E065-544页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110643695A (en) | 2020-01-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2904118B1 (en) | Urine exosome mrnas and methods of using same to detect diabetic nephropathy | |
| US20200208203A1 (en) | Kit for diagnosis of systemic lupus erythematosus and probe for the detection and quantification of the methylation level of an ifi44l fragment | |
| US11339438B2 (en) | Method for assessing the risk of complications in patients with systemic inflammatory response syndrome (sirs) | |
| CN107475388B (en) | Application of nasopharyngeal carcinoma related miRNA as biomarker and nasopharyngeal carcinoma detection kit | |
| CN111826466B (en) | Hepatitis B infected patient or carrier exosome miRNA molecular marker combination and screening kit | |
| CN106755464A (en) | For the method for screening the gene marker of intestinal cancer and/or stomach cancer, the gene marker and application thereof that is screened with the method | |
| CN109609630B (en) | Molecular marker for early gastric cancer diagnosis and application thereof | |
| CN110964823A (en) | DNA methylation kit for colorectal cancer detection and detection method | |
| CN110643695B (en) | Systemic Lupus Erythematosus (SLE) circular RNA marker and detection reagent | |
| CN108866187B (en) | Long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis and application thereof | |
| CN107460241B (en) | Application of exosome small-molecule RNA in acute myocardial infarction risk assessment | |
| CN109536502B (en) | PCR (polymerase chain reaction) internal reference applicable to plasma exosome miRNA of patient with gestational trophoblastic tumor | |
| EP2893354B1 (en) | Use of interleukin-27 as a diagnostic biomarker for bacterial infection in critically ill patients | |
| EP2603604A1 (en) | Method and kit for the diagnosis and/or prognosis of tolerance in liver transplantation | |
| CN111455037B (en) | Coronary heart disease molecular diagnosis marker based on plasma exosome cyclic RNA and application thereof | |
| KR102229647B1 (en) | MiRNA bio-marker for non-invasive differential diagnosis of acute rejection in kidney transplanted patients and uses thereof | |
| CN108998528B (en) | Lung cancer diagnosis molecular marker lncRNA LINC00516, kit and application thereof | |
| WO2012006534A4 (en) | Methods, compositions and kits for diagnosing and treating alzheimer's disease using mitochondrial co3 gene mutations | |
| WO2024036785A1 (en) | Dna methylation marker combination for early screening of gastric cancer and kit | |
| CN110699450A (en) | Application of miRNA biomarker in diagnosis and prognosis of liver disease | |
| CN116064813B (en) | TRIM29 gene methylation detection primer composition, methylation detection method and application | |
| CN116121245B (en) | Small molecule RNA and application thereof in early diagnosis of endometriosis | |
| CN110777200B (en) | GAN gene for early diagnosis of atrial fibrillation | |
| CN110484620B (en) | Biomarker and application thereof in preparation of product for diagnosing PTMC (ptm-associated tumor cell) | |
| CN114507740B (en) | Biomarkers, nucleic acid products and kits for gastrointestinal cancer diagnosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200527 Address after: 200031 building 35, No. 320, Yueyang Road, Xuhui District, Shanghai Applicant after: Center for excellence and innovation of molecular cell science, Chinese Academy of Sciences Applicant after: RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAOTONG University SCHOOL OF MEDICINE Address before: 200031 No. 320, Yueyang Road, Shanghai, Xuhui District Applicant before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES Applicant before: RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAOTONG University SCHOOL OF MEDICINE |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |