WO2022124564A1 - Procédé de prédiction et de diagnostic de neuropathie diabétique à l'aide de micro-arn, et kit associé - Google Patents

Procédé de prédiction et de diagnostic de neuropathie diabétique à l'aide de micro-arn, et kit associé Download PDF

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WO2022124564A1
WO2022124564A1 PCT/KR2021/014992 KR2021014992W WO2022124564A1 WO 2022124564 A1 WO2022124564 A1 WO 2022124564A1 KR 2021014992 W KR2021014992 W KR 2021014992W WO 2022124564 A1 WO2022124564 A1 WO 2022124564A1
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diabetic neuropathy
mir
nucleic acid
predicting
acid sequence
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Korean (ko)
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박영광
임장미
장지련
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주식회사 레피겐엠디
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

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  • the present invention relates to a method for diagnosing diabetic neuropathy and a diagnostic kit using the same.
  • it relates to a method for diagnosing or predicting diabetic neuropathy using microRNA, which can be an indicator of diabetic neuropathy, and a kit for the same. More specifically, it relates to a method and a kit capable of increasing the accuracy of diagnosis of diabetic neuropathy by using microRNAs showing opposite expression levels.
  • Diabetic neuropathy a common microvascular complication of type 1 diabetes mellitus and type 2 diabetes mellitus, is a symptom of peripheral nerve dysfunction in diabetic patients. defined by existence. Population- and clinical-based studies suggest that the prevalence of diabetic neuropathy after 20 years of type 1 diabetes is 20% and increases by about 10% to 15% to 50% at 10 years of type 2 diabetes. Despite these studies, the pathophysiology of diabetic neuropathy has not been clearly defined because of numerous closely related causal mechanisms and difficulties in establishing a definitive diagnostic method.
  • micro-RNA micro-RNA or miRNA, hereinafter abbreviated as 'miRNA'
  • miRNA is an untranslated RNA composed of several to several tens of nucleotides, and performs the functions of RNA silencing in the transcriptional stage and regulating gene expression in the post-transcriptional stage. It is known that the function of these miRNAs is performed through the formation of base pairs with complementary sequences in mRNA (Bartel, D.P., Cell, 136(2): 215-233, 2009).
  • miRNA was first discovered in C. elegans, various types of miRNAs have been reported one after another as it is known that it is being used as a regulatory mechanism for gene expression in animals, plants and viruses, due to mutations in miRNA genes, etc. It is known that certain diseases, such as cancer, may occur due to the dissonance of the functions of : 834-838, 2005).
  • the technical problem to be solved by the present invention is to measure the expression level of miRNA that is over-expressed or under-expressed under diabetic neuropathy and compared with a normal control group to treat diabetic neuropathy. It is to provide a simple method for diagnosing or predicting expression.
  • Another object is to provide a diagnostic kit for diabetic neuropathy using such biomarkers.
  • composition for prediction or diagnosis of diabetic neuropathy for solving any of the above problems is for measuring one or more biomarkers selected from the group consisting of miR-122, miR-199b, and miR-8485 Includes reagents.
  • the reagent may include a nucleic acid sequence of each biomarker, a nucleic acid sequence complementary to the nucleic acid sequence, and a fragment of the nucleic acid sequence.
  • the nucleic acid sequence or fragment of the nucleic acid sequence may be a primer or a probe capable of specific binding to each of the markers, or a primer and a probe.
  • the reagent is a reverse transcription polymerase chain reaction of each biomarker, a polymerase chain reaction, a competitive polymerase chain reaction, a nuclease protection assay (RNase, S1 nuclease assay), an in situ hybridization method, a ligation-based polymerase chain reaction, a micro It may be a reagent used in an array or northern blot.
  • the method for detecting a diabetic neuropathy marker according to an embodiment of the present invention for solving any of the above other problems is a subject suspected of diabetic neuropathy in order to provide information necessary for diagnosing or predicting diabetic neuropathy.
  • the detection may be by a hybridization or nucleic acid amplification method.
  • the hybridization may be a microarray analysis and the nucleic acid amplification may be reverse transcriptase polymerase chain reaction.
  • the biological sample may be whole blood, plasma, or serum from a diabetic neuropathy patient.
  • the method may be to use the average value of the expression levels of the miR-122, miR-199b, and miR-8485 markers.
  • biomarkers selected from the group consisting of miR-122, miR-199b, and miR-8485 for predicting or diagnosing diabetic neuropathy according to an embodiment of the present invention for solving any of the above other problems Reagents for marker measurement are included.
  • diabetic neuropathy can be diagnosed simply by measuring the expression level of miRNAs showing a specific expression pattern under diabetic neuropathy and comparing it with a normal control group.
  • 1 is a heat map analysis result between two groups using a next-generation genomic analysis method for discovering miRNA biomarkers in a normal group and a diabetic neuropathy group.
  • 'comprises' and/or 'comprising' does not exclude the presence or addition of one or more other components in addition to the stated components.
  • Numerical ranges indicated using 'to' indicate numerical ranges including the values stated before and after them as lower and upper limits, respectively.
  • 'About' or 'approximately' means a value or numerical range within 20% of the value or numerical range recited thereafter.
  • the term 'diagnosis' refers to confirming the presence or characteristics of a pathological condition. That is, to determine the susceptibility of the test subject to the disease for a specific disease or disorder, to determine whether or not to currently have the specific disease or disorder, to determine the prognosis of the subject suffering from the specific disease or disorder adjudicating, monitoring the condition of the subject to provide information about whether the disease has relapsed after treatment or therametrics, such as efficacy of the treatment.
  • the diagnosis can be interpreted not only as confirming whether the progress or onset of diabetic neuropathy, but also as confirming the possibility of the onset of diabetic neuropathy.
  • the term 'diagnostic subject' refers to a specimen or biological sample such as blood, body fluid, secretion, tissue, or sample isolated from a patient, individual, or these for diagnosing a corresponding indication.
  • the expression level of miRNA of the present invention can be measured by collecting and detecting blood, body fluid, tissue, nerve cell or nerve tissue of a patient or individual.
  • Biological samples are taken from investigators expected to have diabetic neuropathy.
  • control samples may be taken from investigators already aware of the disease state to corroborate the data obtained.
  • the biological sample refers to an organ, tissue, cell or body fluid derived from an organism.
  • biological samples include, but are not limited to, tissue sections, whole blood, plasma, serum, urine or blood-derived white blood cells, red blood cells, or platelets, or tissue or cell cultures. Also, one or more samples may be mixed and used.
  • the biological sample may be obtained directly from a subject by a routine sample obtaining method from a body having or suspected of having diabetic neuropathy, or may be previously isolated and stored.
  • the sample may be used as a biological sample after purification from the obtained liquid.
  • the expression level of the nucleic acid marker in the present invention is determined in a biological sample derived from a subject.
  • the sample used for detection in the in vitro method of the present invention should generally be collected in a clinically acceptable manner, and is preferably preserved in a nucleic acid (especially RNA) or protein method.
  • a sample awaiting analysis is usually taken from blood.
  • the term 'normal control' refers to a patient, individual, or blood, body fluid, secretion, sample, etc. isolated from a patient or individual who does not have the relevant indication, and the miRNA related to the indication is expressed at a normal level to be diagnosed It can be a criterion for evaluating the miRNA expression level of
  • the term 'biomarker' or 'diagnosis marker' refers to a tissue or cell in which diabetic neuropathy has occurred can be diagnosed by distinguishing it from normal cells or tissues or cells that have received appropriate diabetic neuropathy treatment. It contains non-coding nucleic acids that show an increase or decrease in the diseased tissue or site compared to a normal sample (control).
  • the biomarker according to the present disclosure may be used as one or a combination of two or more, for example, two or three combinations, and may be used together with an existing marker and/or a diagnostic method.
  • 'miRNA (micro-RNA)' refers to a target RNA that promotes degradation. or 21 to 23 non-coding RNAs that post-transcriptionally regulate gene expression by inhibiting their translation.
  • the mature sequence of the miRNA used herein can be obtained from the miRNA database (http://www.mirbase.org).
  • microRNA is transcribed into a precursor of about 70-80 nt (nucleotide) in length with a hairpin structure called pre-miRNA, and then cut by the RNAse III enzyme Dicer to produce a mature form.
  • MicroRNA forms a ribonucleo complex called miRNP to cleave a target gene through complementary binding to a target site or inhibit translation. More than 30% of human miRNA exists as a cluster, and after being transcribed into a single precursor, it undergoes a cleavage process to form a final mature miRNA.
  • miRNAs modified to have a sequence that maintains 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more homology to the miRNA of the present invention. It will be readily understood that it is equivalent to miRNA.
  • nucleic acid includes polynucleotides, oligonucleotides, DNA, RNA, and analogs and derivatives thereof, and includes, for example, peptide nucleic acids (PNA) or mixtures thereof.
  • PNA peptide nucleic acids
  • the nucleic acid may be single or double-stranded, and may encode a molecule including a polypeptide, mRNA, microRNA, or siRNA.
  • the term 'primer' is a nucleic acid sequence having a short free 3' hydroxyl group, which can form a complementary template and base pair, and is a start for template strand copying. It refers to a short nucleic acid sequence that functions as a point. Primers are capable of initiating DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures.
  • reagents for polymerization ie, DNA polymerase or reverse transcriptase
  • the term "complementary" means that under certain hybridization (hybridization) or annealing conditions, preferably under physiological conditions, the antisense oligonucleotide is sufficiently complementary to selectively hybridize to the target of the microRNA according to the present application, , has a meaning encompassing both partially or partially substantially complementary and perfectly complementary, and preferably means completely complementary.
  • substantially complementary refers to a degree of complementarity that is not completely complementary, but is sufficient to bind to a target sequence and produce an effect sufficient to interfere with the effect according to the present application, that is, microRNA activity.
  • the term 'probe' refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to a gene or mRNA, oligonucleotide It may be manufactured in the form of a probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.
  • the present invention is based on the discovery of a non-coding RNA marker such as miRNA that can be used as a biomarker that enables the prediction and accurate early diagnosis of diabetic neuropathy.
  • the present invention relates to a composition for predicting and diagnosing diabetic neuropathy, comprising a reagent for detecting one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485.
  • a person skilled in the art can select a combination of markers satisfying the desired sensitivity and specificity through a method such as an analysis using a biological sample from a subject, including a normal person and a patient, and/or a logistic regression analysis, such as the method described in the Examples herein. will be.
  • a combination of miR-122, miR-199b, and miR-8485 is used.
  • a blood sample such as whole blood, serum or plasma is used.
  • urine, whole blood, serum and/or plasma may be used.
  • a tissue/cell or an in vitro cell culture obtained from a subject having, suspected, or likely to develop diabetic neuropathy may be used, but is not limited thereto. It also includes fractions or derivatives of the blood, cells or tissues. In the case of using cells or tissues, the cells themselves or a lysate of the cells or tissues may be used.
  • a subject herein includes a mammal suspected of having a disease, a mammal who has been treated after having a disease but is suspected of relapse, particularly a human.
  • composition according to the present application detects the expression level of the one or more miRNAs in a biological sample, and compares it with a control or reference group, and can diagnose or predict diabetic neuropathy according to the degree of increase or change in the expression level.
  • composition according to the present application can measure/detect one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485 using the following method, and thus used in such method It may contain reagents that are
  • the measurement of miRNA according to the present disclosure includes methods for qualitative, quantitative and semi-quantitative detection of a desired miRNA. Any known method related to nucleic acid detection, for example, a nucleic acid hybridization and/or polymerization and/or amplification method described below and/or a hybridization-based ligation method may be used.
  • the biomarker according to the present disclosure can be detected at the level of the presence or absence of a nucleic acid, particularly miRNA, and/or its expression level itself, change in expression level, and difference in expression level through quantitative or qualitative analysis.
  • Nucleic acid hybridization can be performed using a nucleic acid biochip array (microarray) or in situ hybridization.
  • miRNA microarray technology enables the analysis of multiple miRNAs simultaneously.
  • Nucleotides complementary to the miRNA according to the present disclosure may be spotted on a coated carrier or may be spotted on a carrier by an in situ synthesis method.
  • miRNA isolated from a biological sample may be detected by incorporation of a label (eg, biotin, fluorescent dye) detected by an enzymatic reaction after hybridization with a complementary sequence on the carrier.
  • the miRNA isolated from the biological sample is labeled with a fluorescent material to bind to the corresponding sequence, and the resulting fluorescent signal is indicative of the presence of a specific miRNA.
  • Microarray fabrication techniques are described, for example, in Schena et al., 1996, Proc Natl Acad Sci USA. 93(20):10614-9; Schena et al., 1995, Science 270(5235):467-70; and U.S. Pat. Nos. 5,599,695, 5,556,752 or 5,631,734.
  • Nucleic acid polymerization or amplification methods may also be used for the detection of miRNAs according to the present application, and are particularly suitable for detecting miRNAs present in trace amounts.
  • Various known nucleic acid amplification or synthesis methods can be used, for example, reverse transcription reaction, reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, PCR, real-time PCR, quantitative RT-PCR, quantitative PCR, NASBA ( Nucleic Acid Sequence-Base Amplification), LCR (Ligase Chain Reaction), Multiple ligatable probe amplification, Invader Technology (Third Wave), SDA (Strand Displacement Amplification), TMA (Transcription Mediated Amplification), and Eberwine RNA It may include, but is not limited to, amplification.
  • a real-time quantitative PCR method is used after the reverse transcription reaction, which may be performed with reference to, for example, Chen et al., Nucleic Acids Research, 33(20):e170, 2005.
  • RT-PCR is a method to isolate a sample RNA, specifically miRNA, synthesize cDNA therefrom, and then use a specific primer or a combination of a primer and a probe to detect a specific gene in the sample, and the presence/ It is a method that can determine the absence or expression level. Such methods are described, for example, in (Han, H. et al, 2002. Cancer Res. 62: 2890-6).
  • a typical PCR method consists of three steps for amplification of a specific target sequence, consisting of denaturation of the template, annealing in which forward and reverse primers bind to the target sequence, and elongation by thermostable polymerase, in several cycles, e.g., usually 20 More than one time is performed. Alternatively, annealing and stretching may be performed in the same step. Since mature miRNA is single-stranded, reverse transcription reaction can be performed first before PCR. The reverse transcription reaction requires the use of a primer and a reverse transcriptase. In PCR and quantitative PCR, one set of primers, forward and reverse, are used.
  • the length of the primer is determined according to various factors such as the hybridization temperature, the composition of the target sequence, and the complexity of the target sequence. In one embodiment, the length of the primer is about 10-35 nucleotides, for example 15, 20, 25, 30 or 35 nucleotides.
  • the forward primer includes at least one sequence capable of specifically binding to the biomarker miRNA, and may further include a non-complementary sequence on the 5' side.
  • the sequence of the reverse primer may be independent of the sequence of the biomarker, and a plurality of miRNA biomarkers may be amplified with one type of reverse primer, or may include one or more sequences specific for the biomarker.
  • two or more miRNAs are amplified in one reaction using multiple quantitative PCR and multiple quantitative RT-PCR methods.
  • one or more pairs of primers and/or probes are used, for example, each pair of primers specifically amplifies a specific miRNA, and the probe is used to distinguish each amplified miRNA to enable multiple amplification.
  • Reverse transcription and PCR can be performed together in reverse transcription quantitative PCR.
  • the reaction includes both reverse transcriptase and thermostable polymerase, and a "hot start" reaction method that controls the activity of thermostable polymerase by chemical or thermal method (See, eg, US Pat. Nos. 5,411,876, 5,550,044, etc.) may be used.
  • the amplified product has a sequence corresponding to the molecule used as a template, and can be analyzed by various methods known in the art. Such methods are known in the art, for example, gel electrophoresis, real-time PCR analysis, single strand conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP), capillary zone electrophoresis (CZE), WAVE (HPLC-based nucleic acid) analyzing technology), including, but not limited to, microchips.
  • SSCP single strand conformational polymorphism
  • RFLP restriction fragment length polymorphism
  • CZE capillary zone electrophoresis
  • WAVE HPLC-based nucleic acid
  • hybridization-based ligation techniques can be used for quantitative analysis of miRNAs. Such methods are known in the art and do not bind a detectable probe that binds to a target nucleic acid sequence, such as, for example, oligonucleotide ligation (OLA) and methods using HARP-like probes described in US Publication 2006-0078894. It includes, but is not limited to, a method for isolating from a non-existing probe.
  • OLA oligonucleotide ligation
  • MLPA Multiplex Ligation-dependent Probe Amplification
  • the miRNA after hybridization, amplification and/or hybridization-based ligation reaction as described above can detect hybridization or amplified miRNA products through, for example, staining or labeling of a target, staining or labeling of a primer or probe.
  • a technique known in the art may be used, and a person skilled in the art will be able to select an appropriate method in consideration of the sensitivity of detection and/or the amount of the target. Depending on the sensitivity of the detection method and/or the amount of target, amplification may not be necessary prior to detection.
  • miRNAs can be detected by direct or indirect methods.
  • miRNA is labeled with a detectable label bound thereto, and then bound to a probe connected to a solid support such as a bead, and then detected by screening the labeled miRNA.
  • a labeled probe may be used for direct detection, and is detected through screening of the labeled probe after specific binding to the miRNA.
  • the amplified miRNA is detected using a bead conjugated to a probe capable of capturing a desired nucleic acid.
  • the probe may be labeled with a fluorescent material.
  • Labels for detection include, but are not limited to, compounds capable of generating or canceling a detectable fluorescence, chemiluminescent or bioluminescent signal such as a light emitting, light scattering, light absorbing material, for example, Garman Reference may be made to A., Non-Radioactive Labeling, Academic Press 1997.
  • Fluorescent materials include, but are not limited to, fluorescein (eg, US Pat. No. 6,020,481), rhodamine (eg, US Pat. No. 6,191,278), benzophenoxazine (eg, US Pat. No. 6,140,500), donors and acceptors.
  • Energy transfer fluorescent dyes including (eg US Pat. No.
  • SYBR-Green, 6-carboxyfluorescein (“FAM”), TET, ROX, VICTM, or JOE is used as the fluorescent label.
  • FAM 6-carboxyfluorescein
  • TET tetrachloride
  • ROX X-Reactive OLED
  • VICTM VICTM
  • JOE JOE
  • a probe labeled with two fluorescent substances, a reporter fluorescent material and an erasing fluorescent material is used.
  • a fluorescent material emitting a spectrum with a distinguishable wavelength is used as the fluorescent material.
  • the marker is a compound capable of enhancing, stabilizing, or affecting the binding of a nucleic acid
  • an intercalator including ethyl bromide and SYBR-Green, a minor groove binder, and a crosslinkable functional group can be used, but is not limited thereto, and is described in Blackburn et al., eds. See “DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996).
  • composition according to the present disclosure may include reagents used in any one or more of the methods described above.
  • a probe and/or primer pair specific for the mRNA of the present marker is included.
  • “Primer” or “probe” means a nucleic acid sequence having a free 3' hydroxyl group capable of complementary binding to a template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template do.
  • the reagent may be labeled with a chromogenic, luminescent or fluorescent substance as described above for detection of the amplified product.
  • reverse transcription PCR polymerase chain reaction
  • the detection reagent may be provided in the form of an array or chip including a microarray. Detection reagents may be labeled directly or indirectly in a sandwich form for detection. In the case of the direct labeling method, serum samples used for arrays and the like are labeled with a fluorescent label such as Cy3 or Cy5.
  • biomarker according to the present disclosure or a composition comprising the same may be usefully used for diagnosis, prediction and/or prognosis measurement of diabetic neuropathy.
  • composition herein, a kit comprising said composition, or a method.
  • the kit of the present application is used for nucleic acid amplification, in particular for amplification using RT-PCR.
  • the kit contains the necessary buffer for the reaction of RT-PCR, reverse transcriptase, Taq polymerase and MgCl2.
  • Various buffers known in the art may be used, for example, Tris-HCl, pH 9.0 buffer may be used, but is not limited thereto.
  • Reverse transcriptase and Taq polymerase are commercially available, for example, AmpliTaq Gold (Applied Biosystems, USA) capable of hot start reaction with polymerase may be used, and an appropriate concentration, for example, 1.5 mM to 2.5 mM MgCl2 may be included.
  • the kit according to the present application further comprises a positive control group, a negative control group and instructions for use.
  • the negative control group may include a sample that does not contain miRNA, and the positive control group may include one or more of the detection target miRNAs.
  • the present application also provides a sample derived from a subject suspected of diabetic neuropathy in order to provide information necessary for the diagnosis or prognosis of diabetic neuropathy; measuring the expression level of one or more markers selected from the group consisting of miR-122, miR-199b, and miR-8485 in the sample; comparing the measurement result with the corresponding result of the corresponding marker in the control group; and when there is a change in the expression level of the subject sample compared to the control sample, determining it as diabetic neuropathy.
  • Reagents and the like used in the method of the present application may be referred to as previously described.
  • a sample from a diabetic patient may be used for prediction or early diagnosis of diabetic neuropathy.
  • the method may be performed over a specific period of time, for example, several times over a year or once a year, and may be used to monitor changes in expression patterns.
  • an increase or decrease in expression can be associated with a diabetic neuropathy state. It can be used to determine the onset, progression, exacerbation, etc. of diabetic neuropathy by comparison with a previous test value for the same subject or a control value.
  • Prophylactic measures can be taken to prevent the progression or onset of diabetic neuropathy based on changes in diabetic neuropathy markers over time.
  • biomarkers can be used as an auxiliary means, and other diagnostic methods such as biopsy, ultrasound, computerized axial tomography (CT scan) or magnetic resonance imaging (magnetic resonance) imaging (MRI)).
  • CT scan computerized axial tomography
  • MRI magnetic resonance imaging
  • the present invention can be practiced using conventional techniques within the skill level of those skilled in the art in cell biology, cell culture, molecular biology, gene transformation technology, microbiology, and DNA recombination technology.
  • RNA was isolated from the obtained serum using the Qiagen RNA extraction kit according to the manufacturer's method, and the quality of the isolated total RNA was checked using an Agilent 2100 Bioanalyzer according to the manufacturer's method.
  • Vitinylated cDNA was synthesized from the total RNA isolated in Example 1 using SMARTer smRNA seq kit for Illumina (Takara). The cDNA was then hybridized to Affymetrix GeneChip ® miRNA 4.0 according to the manufacturer's standard method. Measurement and analysis of fluorescence intensity after hybridization were performed using Genechip operating software (Affymetrix) according to the manufacturer's method. A total of 6658 human miRNAs were compared and analyzed in 3 cases of normal persons and diabetic neuropathy, respectively, and as shown in FIG. 1 , a heat map for miRNAs related to diabetic neuropathy (yellow: overexpressed miRNAs more than twice, blue) : Two-fold or more underexpressed miRNAs).
  • RNA sample 10 pM stem-loop RT primer, 10X RT buffer, 5 U/uL poly A polymerase, 2 mM of each dNTP, 200 U/uL M-MLV, 10mM ATP, and 5 U/uL RNase Inhibitor (enzynomics, Korea) was used to synthesize cDNA by reverse transcription. Then cDNA was denatured for 5 min at 95 °C using respective miRNA primers and SYBR greenI. Amplification was performed by 40 cycles of 10 seconds at 95 degrees and 40 seconds at 60 degrees, and the signals were collected and analyzed in real time with Bio-Rad CFX-Dx 3.1.

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Abstract

La présente invention concerne les éléments suivants : un procédé pour diagnostiquer ou prédire la neuropathie diabétique en utilisant un micro-ARN, pouvant être un indicateur de la neuropathie diabétique ; et un kit pour le procédé. Une composition pour prédire ou diagnostiquer la neuropathie diabétique comprend au moins un réactif de mesure de biomarqueur choisi dans le groupe constitué par miR-122, miR-199b, et miR-8485.
PCT/KR2021/014992 2020-12-10 2021-10-25 Procédé de prédiction et de diagnostic de neuropathie diabétique à l'aide de micro-arn, et kit associé WO2022124564A1 (fr)

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