CN113817819B - Application of LINC01996 in diagnosis of allergic airway inflammation - Google Patents

Application of LINC01996 in diagnosis of allergic airway inflammation Download PDF

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CN113817819B
CN113817819B CN202111230526.5A CN202111230526A CN113817819B CN 113817819 B CN113817819 B CN 113817819B CN 202111230526 A CN202111230526 A CN 202111230526A CN 113817819 B CN113817819 B CN 113817819B
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毛正道
刘志光
张倩
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Abstract

The invention discloses application of LINC01996 in diagnosis of allergic airway inflammation. By detecting the expression level of LINC01996, allergic airway inflammation can be rapidly and accurately diagnosed. The LINC01996 serving as a marker can improve the sensitivity and specificity of diagnosis of allergic airway inflammation, and has important clinical significance and application prospect.

Description

Application of LINC01996 in diagnosis of allergic airway inflammation
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of LINC01996 in diagnosis of allergic airway inflammation.
Background
Allergic Rhinitis-Asthma Syndrome (CARAS) is a new medical diagnostic concept proposed by World Allergy Organization (WAO), and refers to the concurrent, clinical or sub-clinical symptoms of chronic Allergic inflammation and hyperresponsiveness caused by upper airway Allergy (Allergic Rhinitis, AR) and lower airway Allergy (bronchial Asthma, BA). AR is an allergic disease occurring in the nasal mucosa and is manifested by nasal secretion hyperfunction, nasal mucosa swelling, watery nasal discharge, nasal obstruction caused by catarrhal inflammation, nasal itching and sneezing caused by high reactivity. BA is manifested by airway desquamation, hyperresponsiveness (AHR), and wheezing, shortness of breath, chest tightness, and coughing, among others, due to restricted airflow. Some CARAS is associated with allergic conjunctivitis with symptoms such as itching of the eye, tearing, and the like. Often the nasal manifestations are exacerbated in the morning and the asthma symptoms are exacerbated at night. CARAS is a disease process which is induced by a plurality of factors and has an acute onset when the respiratory system diseases are aggravated, can suddenly occur, can be automatically relieved or relieved after treatment, is susceptible to repeated attacks caused by certain internal and external factors, and has obvious negative effects on health and life quality. CARAS is a common disease and a frequently-occurring disease of a respiratory system, can be developed at any age at all seasons, and has an increasing incidence rate year by year due to risk factors such as environmental factors, occupational exposure dust, poor dietary habits, drinking, smoking and drug allergy besides genetic factors, thereby attracting attention of the medical community. At present, CARAS adopts a combined diagnosis mode of allergic rhinitis and asthma in diagnosis. There are few reports on the research on biomarkers that can be used to diagnose allergic rhinitis-asthma syndrome.
Disclosure of Invention
The present invention aims to provide a marker useful for diagnosing allergic rhinitis-asthma syndrome, which addresses the deficiencies of the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides the use of an agent for measuring the level of expression of a biomarker in a biological sample, said biomarker comprising LINC01996, in the manufacture of a product for diagnosing allergic airway inflammation in a subject.
In certain embodiments, the allergic airway inflammation comprises allergic rhinitis, asthma, allergic rhinitis-asthma syndrome. As a preferred embodiment, the allergic airway inflammation is allergic rhinitis-asthma syndrome.
In one embodiment, a decreased expression level of LINC01996 as compared to a reference value range for the biomarker in a non-diseased control subject is indicative that the subject has allergic airway inflammation.
In another aspect, the invention provides a method for diagnosing allergic airway inflammation in a subject. The method comprises a) measuring the level of LINC01996 in a biological sample derived from the subject; and b) analyzing the level of LINC01996 in combination with a corresponding reference value range of LINC01996, wherein a differential expression of LINC01996 in the biological sample compared to the reference value range of LINC01996 of a non-diseased control subject is indicative of the subject having allergic airway inflammation. The reference value range may represent the level of LINC01996 found in one or more samples of one or more subjects (e.g., healthy subjects) who do not suffer from allergic airway inflammation. Alternatively, the reference value may represent the level of LINC01996 found in one or more samples of one or more subjects with allergic airway inflammation.
In the present invention, the expression level of LINC01996 can be detected by microarray analysis, polymerase Chain Reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), northern blot, southern blot or Serial Analysis of Gene Expression (SAGE).
In certain embodiments, the biological sample is blood.
In certain embodiments, the reagents include primers, probes, microarrays.
In one embodiment, the probe comprises a fluorescent label, a bioluminescent label, a chemiluminescent label, a colorimetric label, or an isotopic label.
In another aspect, the invention also provides a product for diagnosing allergic airway inflammation in a subject, the product comprising an agent for detecting the expression level of LINC01996 in a biological sample.
In certain embodiments, the allergic airway inflammation comprises allergic rhinitis, asthma, allergic rhinitis-asthma syndrome. As a preferred embodiment, the allergic airway inflammation is allergic rhinitis-asthma syndrome.
In certain embodiments, the product comprises a kit, chip, strip.
In one embodiment, the kit comprises information in electronic or paper form comprising instructions to correlate the detected level of LINC01996 with the allergic airway inflammation.
In another aspect, the present invention also provides a diagnostic system for performing a computer-implemented method for diagnosing a patient suspected of having an allergic airway inflammation, the computer performing steps comprising:
1) Accepting the entered patient data comprising an expression level value of LINC01996 in a biological sample from the patient;
2) Analyzing the level of the biomarker and comparing to a corresponding reference value range for the biomarker, if the level of LINC01996 of the patient is lower than the reference value range for a control subject that is not diseased, indicating that the patient has allergic airway inflammation;
3) Displaying diagnostic information about the patient,
wherein the diagnostic system comprises:
1) A storage component for storing data, wherein the storage component has instructions for determining a diagnosis of a subject stored therein;
2) A computer processor for processing data, wherein the computer processor is coupled to the storage component and configured to execute instructions stored in the storage component in order to receive patient data and analyze the patient data according to one or more algorithms; and
3) A display assembly for displaying diagnostic information about the patient.
In certain embodiments, the allergic airway inflammation comprises allergic rhinitis, asthma, allergic rhinitis-asthma syndrome. As a preferred embodiment, the allergic airway inflammation is allergic rhinitis-asthma syndrome.
Drawings
FIG. 1 is an electrophoretogram of RNA;
FIG. 2 is a graph of real-time amplification and product dissolution of an internal reference GAPDH gene, wherein A is a graph of real-time amplification of the GAPDH gene and B is a graph of product dissolution;
FIG. 3 is a LINC01996 gene real-time amplification curve graph and a product dissolution curve graph, wherein a is a LINC01996 gene real-time amplification curve graph and B is a product dissolution curve graph;
FIG. 4 is a bar graph of LINC01996 differential expression;
FIG. 5 is a ROC plot of LINC01996.
Detailed Description
The practice of the present invention will employ, unless otherwise indicated, conventional methods of pharmacology, chemistry, biochemistry, recombinant DNA techniques and immunology, which are within the skill of the art. These techniques are explained fully in the following documents: handbook of Experimental Immunology, volumes I-IV (D.M.Weir and C.C.Blackwell, blackwell Scientific Publications); l. lehninger, "Biochemistry (Biochemistry) (Worth Publishers, current edition); sambrook et al, molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual) (3 rd edition, 2001); methods in enzymology (Methods in enzymology), eds (s.colowick and n.kaplan, academic Press, inc.).
1. Definition of
In describing the present invention, the following terminology will be employed and is intended to be defined as indicated below.
In the context of the present invention, a "biomarker" refers to a biological compound, such as a polynucleotide, that is differentially expressed in a sample obtained from a patient having allergic airway inflammation as compared to a comparable sample obtained from a control subject (e.g., a person who is negative in diagnosis, a normal or healthy subject, or a subject who is not diseased). A biomarker may be a nucleic acid, nucleic acid fragment, polynucleotide, or oligonucleotide that is detectable and/or quantitative.
The terms "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" are used herein to include nucleotides (ribonucleotides or deoxyribonucleotides) of any length in the form of a polymer. This term refers only to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded and single-stranded DNA as well as triple-stranded, double-stranded and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide. More specifically, the terms "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide (which is an N-or C-glycoside of a purine or pyrimidine base). There is no intended length distinction between the terms "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule", and these terms are used interchangeably.
A polynucleotide is differentially expressed between two samples if the amount of the polynucleotide in one sample is statistically significantly different from the amount of the polynucleotide in the other sample. For example, a polynucleotide is differentially expressed in two samples if it is present in an amount of at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% of the amount it is present in the other sample, or if it is detectable in one sample and not detectable in the other sample.
The terms "subject", "individual" and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, prognosis, treatment or therapy. Other subjects may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on. In some cases, the methods of the invention find application in laboratory animals, veterinary applications, and in the development of animal disease models, including (but not limited to) rodents, including mice, rats, and hamsters, and primates.
As used herein, "biological sample" refers to a sample of tissue, cells, or fluid isolated from a subject, including, but not limited to, samples such as blood, buffy coat, plasma, serum, blood cells (e.g., peripheral Blood Mononuclear Cells (PBMCs), rod shaped nuclear cells, neutrophils, monocytes, or T cells), fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, skin samples, external secretions of the skin, respiratory tract, intestinal tract, and genitourinary tract, tears, saliva, milk, organs, biopsies, and in vitro cell culture components, including, but not limited to, conditioned media resulting from the growth of cells and tissue in culture, such as recombinant cells and cell components. In a specific embodiment of the present invention, the biological sample is blood.
As used herein, "diagnosis" generally includes a determination as to whether a subject is likely to suffer from a given disease, disorder, or dysfunction. One of skill in the art typically diagnoses based on one or more diagnostic indicators (i.e., biomarkers), the presence, absence, or amount of which is indicative of the presence or absence of a disease, disorder, or dysfunction.
As used herein, a "primer" refers to an oligonucleotide that can be used in an amplification method, such as Polymerase Chain Reaction (PCR), to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a gene of interest (e.g., a LINC01996 sequence or a portion thereof). Typically, at least one PCR primer used to amplify a polynucleotide sequence is sequence specific for the polynucleotide sequence. The exact length of the primer depends on a variety of factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, oligonucleotide primers typically contain at least 10, or 15, or 20, or 25 or more nucleotides, but they may contain fewer or more nucleotides, depending on the complexity of the target sequence. Factors involved in determining the appropriate length of a primer are well known to those skilled in the art. Specific embodiments of primers used are shown in Table 4 herein, in which their specific applications are indicated. In this context, the term "primer pair" denotes a primer pair which hybridizes to the opposite strand of a target DNA molecule or to a region of the target DNA flanking the nucleotide sequence to be amplified. As used herein, the term "primer site" refers to a region of a target DNA or other nucleic acid to which a primer hybridizes.
As used herein, the term "probe" refers to any molecule capable of selectively binding to a particular desired target biomolecule. In some embodiments, the term "probe" herein refers to any molecule that can bind to or be associated with any substrate and/or reaction product and/or protease disclosed herein, either indirectly or directly, covalently or non-covalently, and which association or binding can be detected using the methods disclosed herein. In some embodiments, the probe is a fluorescent probe, an antibody, or an absorbance-based probe. In the case of absorbance-based probes, the chromophore pNA (p-nitroaniline) can be used as a probe for detecting and/or quantifying the target nucleic acid sequence disclosed herein. In some embodiments, a probe may be a nucleic acid sequence comprising a fluorescent molecule or substrate that becomes fluorescent upon exposure to an enzyme, and the nucleic acid sequence is complementary to a fragment of one of the nucleic acid sequences.
2. Modes for carrying out the invention
Before the present invention is described in detail, it is to be understood that this invention is not limited to particular formulations or process parameters, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
The present invention relates to a marker for diagnosing allergic rhinitis-asthma syndrome and application thereof. In particular, the present inventors have found a biomarker, the expression profile of which is useful in diagnosing allergic airway inflammation.
Biomarkers
Biomarkers useful in the practice of the present invention include polynucleotides derived from the nucleotide sequence of a gene or RNA transcript of a gene, including (but not limited to) LINC01996. Differential expression of biomarkers is associated with allergic airway inflammation and therefore, the expression profile of biomarkers can be used to diagnose allergic airway inflammation.
In the present invention, biomarkers such as LINC01996 (gene ID: 400568) include gene and mutations thereof. The term encompasses naturally occurring variants (e.g., splice variants) of the biomarker.
Detecting and measuring biomarkers
In the present invention, the biomarker may be measured by any suitable method known in the art. The measurement of the expression level of the biomarker may be direct or indirect. For example, the abundance level of RNA or protein can be quantified directly. Alternatively, the amount of a biomarker can be determined indirectly by measuring the abundance level of cDNA, amplified RNA or DNA, or by measuring the amount or activity of RNA or other molecules (e.g., metabolites) indicative of the expression level of the biomarker.
In one embodiment, the expression level of the biomarker is determined by measuring the polynucleotide level of the biomarker. Transcript levels of a particular biomarker gene may be determined from the amount of mRNA or polynucleotide derived therefrom present in the biological sample. Polynucleotides can be detected and quantified by a variety of methods including, but not limited to, microarray analysis, polymerase Chain Reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), northern blotting, southern blotting, and Serial Analysis of Gene Expression (SAGE). See, e.g., draghicii "tools for data analysis of DNA microarrays (DataAnalysis tools for DNAmicroarray"), chapman and Hall/CRC,2003; simon et al Design and Analysis of DNA Microarray Studies (Design and Analysis of DNA Microarray investments), springer,2004; real-time PCR: current Technology and applications (Real-Time PCR: current technologies and applications), login, edwards and Saunders, catalog Academic Press,2009; burtin "quantitative PCR A-Z (A-Z of quantitative PCR) in IUL Biotechnology, 5 th), international university Line,2004; velculescu et al (1995) science 270; matsumura et al (2005) cytology (cell. Microbiol.) 7; serial Analysis of Gene Expression (SAGE): methods and protocols (Methods in Molecular Biology) (Serial Analysis of Gene Expression (SAGE): methods and protocols (Methods in Molecular Biology)), I-Press, humana Press,2008; which is incorporated herein by reference in its entirety.
Kit and microarray
The present invention provides a kit for diagnosing allergic airway inflammation, wherein the kit can be used for detecting the biomarker of the present invention. The kit may include one or more reagents for detecting a biomarker, a container for holding a biological sample isolated from a human subject suspected of having allergic airway inflammation; and printed instructions for reacting the reagent with the biological sample or a portion of the biological sample to detect the presence or amount of at least one allergic airway inflammation biomarker in the biological sample. The reagents may be packaged in separate containers.
The kit may comprise one or more containers for holding the compositions contained in the kit. The composition may be in liquid form or may be lyophilized. Suitable containers for the composition include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials, including glass or plastic. The kit may further comprise a package insert containing written instructions for a method of diagnosing allergic airway inflammation.
Microarrays are prepared by selecting probes comprising polynucleotide sequences and then immobilizing such probes on a solid support or surface. For example, the probe may comprise a DNA sequence, an RNA sequence, or a copolymer sequence of DNA and RNA. The polynucleotide sequence of the probe may further comprise DNA and/or RNA analogs or combinations thereof. For example, the polynucleotide sequence of a probe may be a complete or partial fragment of genomic DNA. The polynucleotide sequence of the probe may also be a synthetic nucleotide sequence, such as a synthetic oligonucleotide sequence. The probe sequence may be synthesized enzymatically in vivo, enzymatically in vitro (e.g., by PCR), or non-enzymatically in vitro.
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1Real Time PCR detection of changes in expression levels of target genes in blood samples from patients with allergic rhinitis-asthma syndrome
1. Purpose of experiment
The method is used for detecting the change condition of the transcription level of the target gene lncRNA in the blood sample of the patient with the allergic rhinitis-asthma syndrome by a SYBR Green I Real Time PCR method.
2. Experimental materials
1. Sample List
20 patients with allergic rhinitis-asthma syndrome and 20 normal persons were recruited in the study, clinical information is shown in Table 1, and blood samples were collected for analytical study.
TABLE 1 basic clinical information of the persons participating in this study
Figure BDA0003315669430000091
2. Experiment main reagent
TABLE 2 list of reagents used
Figure BDA0003315669430000092
3. Main experimental instrument
TABLE 3 Instrument List used
Figure BDA0003315669430000093
Figure BDA0003315669430000101
3. Experimental methods
1. Primer design
1.1 Real Time PCR detection of target gene primers. The following primers were synthesized by Bomaide.
TABLE 4 primer sequences
Figure BDA0003315669430000102
2. Procedure of experiment
2.1 extraction of Total RNA from samples
(1) 0.75mL of lysis solution RLS was added per 0.25mL of fluid sample (serum, plasma, cerebrospinal fluid, etc.) and the fluid sample was blown several times with a sample gun to help lyse the cells in the sample. Each 5-10 × 10 6 At least 0.75ml of lysis buffer RLS was added to each cell. The final volume ratio of lysate RLS and liquid sample was always 3:1.
(2) 0.75mL of lysis buffer RLS was added to the EP tube, 0.25mL of blood sample was added, the tube was shaken vigorously for 30 seconds, mixed well, and incubated at 15-30 ℃ for 10min to completely decompose the nucleoproteins.
(3) 0.2mL of chloroform was added to 0.75mL of lysis solution RLS, shaken vigorously for 15s and left at room temperature for 5min.
(4) After centrifugation at 12000rpm for 10min at 4 ℃, the sample will separate into three layers: the lower organic phase, the middle layer and the upper colorless aqueous phase, and RNA is present in the upper aqueous phase. The volume of the aqueous layer was about 70% of the volume of RLS added and the aqueous layer was transferred to a fresh tube for further processing.
(5) 1-fold volume of 70% ethanol was added (please first check if absolute ethanol had been added!), and the mixture was mixed by inversion (precipitation may occur). The resulting solution, together with possible precipitates, is transferred to an adsorption column RA (which is fitted in a collection tube).
(6) Centrifuging at 12000rpm for 45s, discarding waste liquid, and sleeving the adsorption column back to the collection tube again.
(7) Adding 0.5mL of deproteinized solution RE, centrifuging at 12000rpm for 45s, and discarding the waste liquid.
(8) 0.5mL of the rinse solution RW (please first check if absolute ethanol had been added!) was added and centrifuged at 12000rpm for 45s, discarding the waste solution.
(9) 0.5mL of rinsing solution RW was added, and centrifuged at 12000rpm for 45 seconds, and the waste solution was discarded.
And (3) putting the adsorption column RA back into the pore collection tube, centrifuging at 13000rpm for 2min, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction.
Figure BDA0003315669430000111
Taking out the adsorption column RA, placing into an RNase free centrifuge tube, adding 30-50uL RNase free water (better heating effect in water bath at 65-70 deg.C) at the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 2min, and centrifuging at 12000rpm for 1min. If more RNA is needed, the obtained solution can be added into the centrifugal adsorption column again and centrifuged for 1min, or 30ul RNase free water is added additionally and centrifuged for 1min, and the two eluates are combined.
2.2 Synthesis of lncRNAcDNA by reverse transcription
Using the FastQuant cDNA first strand synthesis kit (cat # KR 106) for IncRNA reverse transcription, genomic DNA removal reaction was first performed, 5 Xg DNA Buffer 2.0ul, totalRNA 1ug, and RNase Free ddH were added to the test tube 2 O to make the total volume to 10ul, and water bathHeating at 42 deg.C for 3min, and mixing 10 Xfast RT Buffer 2.0uL, RT Enzyme Mix 1.0uL, FQ-RT Primer Mix2.0uL, RNase Free ddH 2 O5.0 uL, mixing, adding into the test tube, mixing together for 20uL, heating in water bath at 42 deg.C for 15min and 95 deg.C for 3min, and storing at-20 deg.C or lower when the synthesized cDNA is required to be stored for a long time.
2.3 Fluorescent quantitative detection of mRNA
2.3.1 Instrument and analytical method
Using ABI 7300 type fluorescence quantitative PCR instrument and adopting 2- △△CT The method performs a relatively quantitative analysis of the data.
2.3.2 the procedure is as follows:
reaction system (one): amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP 205) and the experimental procedures were performed according to the product instructions. The RealTime reaction system is:
TABLE 5 RealTime reaction System
Reagent Amount of the composition used
2×SuperReal PreMix Plus 10μl
Upstream primer (10 uM) 0.6μl
Downstream primer (10 uM) 0.6μl
50×ROX Reference Dye △ 2μl
DNA template 2ul
Sterilized distilled water 4.8ul
(II) the amplification procedure is as follows: 95 ℃ 15min, (95 ℃ 10sec,55 ℃ 30sec,72 ℃ 32 sec). Times.40 cycles, 95 ℃ 15sec,60 ℃ 60sec,95 ℃ 15 sec).
(III) primer screening
Mixing cDNA of each sample, performing 10-fold gradient dilution by taking the cDNA as a template, taking 2 mu l of each diluted sample as the template, respectively amplifying by using a target gene primer and an internal reference gene primer, simultaneously performing melting curve analysis at 60-95 ℃, and performing primer screening according to the principle of high amplification efficiency and single peak of the melting curve.
(IV) sample RealTimePCR detection
After 10-fold dilution of each sample cDNA, 2. Mu.l of each sample cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification, respectively (see Table five). At the same time, the dissolution curve analysis is carried out at 60-95 ℃.
TABLE 6 sample RealTimePCR detection design
Form panel Sample cDNA Sample cDNA
Repeatedly detecting the number of channels 3 3
Primer and method for producing the same Target gene primer Internal reference gene primer
(V) data statistics
Sorting original result ct values derived by a running program downloading machine according to a sample loading sequence to obtain three multi-hole original ct values of each gene of each sample, respectively calculating the average value of the three multi-hole ct values of a target gene and an internal reference gene in excel, respectively calculating the expression of the target gene relative to the internal reference gene in a control group (cancer adjacent tissues) and a test group (stomach cancer tissues), carrying out statistical analysis by adopting GraphPad software, and adopting t test on the difference between the two.
4. Results of the experiment
1.RNA concentration detection result and 1.5% agarose RNA electrophoresis detection result
TABLE 7 RNA concentration and purity results
Figure BDA0003315669430000131
Figure BDA0003315669430000141
Note:
the RNA dissolved in water results in a low A260/280 ratio
A, the concentration does not reach the standard; b, unqualified A260/A280; c, unqualified electrophoretogram; h, qualified sample evaluation standard:
(1) The concentration is more than 30ng/ul
(2)1.8<A260/A280<2.0
(3) The electrophoretic pattern shows three distinct bands (the third band may not be visible)
TABLE 8 electrophoretic Loading
Figure BDA0003315669430000142
Figure BDA0003315669430000151
Figure BDA0003315669430000161
The RNA electrophoretogram is shown in FIG. 1, wherein M represents DNA Marker: DM2000, from bottom to top, is 100,250,500,750,1000 and 2000bp, wherein 750bp is a bright band.
2. Results and analysis of RealTimePCR detection for each sample
The real-time amplification curve and the dissolution curve of the sample amplification product of each sample are shown in FIGS. 2 and 3.
The statistical results are shown in fig. 4, and LINC01996 expression was down-regulated in the sample of the allergic rhinitis-asthma syndrome group compared to the healthy control group.
Example 2 diagnostic potency validation of LINC01996
SPSS software is used to draw a Receiver Operating Curve (ROC), AUC values, sensitivity and specificity are analyzed, and the individual diagnostic efficacy of the index is judged.
As shown in table 9 and fig. 5, LINC01996 had high diagnostic potency (AUC value of 0.858, sensitivity value of 0.750, specificity value of 0.850), suggesting that LINC01996 can be used for diagnosing allergic rhinitis-asthma syndrome.
TABLE 9 region under ROC Curve
Figure BDA0003315669430000171
The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.
In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.
Sequence listing
<110> second people hospital in Changzhou city
Application of <120> LINC01996 in diagnosis of allergic airway inflammation
<141> 2021-10-22
<160> 8
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Claims (9)

1. Use of an agent for measuring the level of expression of a biomarker in a biological sample, wherein the biomarker comprises LINC01996, in the manufacture of a product for diagnosing allergic rhinitis-asthma syndrome in a subject.
2. The use of claim 1, wherein a decreased expression level of LINC01996 as compared to a reference value range for a biomarker in a non-diseased control subject is indicative of the subject having allergic rhinitis-asthma syndrome.
3. The use according to claim 1, wherein the reagents comprise reagents for measuring the expression level of the biomarker by microarray analysis, polymerase chain reaction, reverse transcriptase polymerase chain reaction, northern blot, southern blot or serial analysis of gene expression.
4. The use according to claim 1, wherein the biological sample is blood.
5. The use according to any one of claims 1 to 4, wherein the reagents comprise primers, probes, microarrays.
6. The use of claim 5, wherein said probe comprises a fluorescent label, a bioluminescent label, a chemiluminescent label, a colorimetric label or an isotopic label.
7. The use according to claim 1, wherein said product comprises a kit, chip, strip.
8. The article of claim 7, wherein said kit comprises information in electronic or paper form, said information comprising instructions relating the level of detection of LINC01996 to said allergic rhinitis-asthma syndrome.
9. A diagnostic system for performing a computer-implemented method for diagnosing a patient suspected of having allergic rhinitis-asthma syndrome, said computer performing steps comprising:
1) Accepting the entered patient data comprising an expression level value of LINC01996 in a biological sample from the patient;
2) Analyzing the level of the biomarker and comparing to a corresponding reference value range for the biomarker, if the level of LINC01996 of the patient is lower than the reference value range for a non-diseased control subject, indicating that the patient has allergic rhinitis-asthma syndrome;
3) Displaying diagnostic information about the patient,
wherein the diagnostic system comprises:
1) A storage component for storing data, wherein the storage component has instructions for determining a diagnosis of a subject stored therein;
2) A computer processor for processing data, wherein the computer processor is coupled to the storage component and configured to execute instructions stored in the storage component in order to receive patient data and analyze the patient data according to one or more algorithms; and
3) A display assembly for displaying diagnostic information about the patient.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107365859A (en) * 2017-08-22 2017-11-21 固安博健生物技术有限公司 Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107365859A (en) * 2017-08-22 2017-11-21 固安博健生物技术有限公司 Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
肺腺癌差异表达基因的分析;姜月;《内蒙古大学硕士毕业论文》;20190418;全文 *

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