KR101063247B1 - Liver tissue specific gene MML2 - Google Patents

Abstract

The present invention provides a microarray for diagnosing a liver-related disease including MML2 (Mus musculus Liver 2) protein, a gene encoding the protein, the gene or an antibody against the protein, which is specifically expressed in liver tissue of a mouse, It relates to a primer set for diagnosing a liver-related disease consisting of primers for the gene, a kit for diagnosing a liver-related disease comprising the primer set, and a method for diagnosing liver-related disease in a mammal using the primer set. .

Liver Specific Genes, MML2 Proteins, Microarrays, Primer Sets, Kits

Description

Liver specific gene MML2

The present invention relates to a liver tissue-specific gene MML2 involved in liver function, and more particularly to MML2 protein, a gene encoding the protein, the gene or an antibody against the protein, which are specifically expressed in liver tissue of a mouse. Using a microarray for diagnosing a liver-related disease comprising, a primer set for diagnosing a liver-related disease consisting of primers for the gene, a kit for diagnosing a liver-related disease comprising the primer set, and the primer set The present invention relates to a method of diagnosing a liver disease of a mammal.

The liver plays a central role in metabolism and has numerous functions such as carbohydrate storage, red blood cell breakdown, protein synthesis, and detoxification. To this end, the liver consists of specialized cellular tissues and secretes unique enzymes, signaling agents, and regulators. Tissue-specific genes can be used as markers for diagnosing diseases by tracking their changes, or they can be themselves targets for the treatment of diseases, or they can act as mediators to help drugs act on specific tissues.

Therefore, the present inventors, while studying the liver specific gene, revealed that the MML2 gene is specifically expressed in liver tissue, and completed the present invention.

In order to solve the above problems, the present invention provides MML2 protein specifically expressed in liver tissue.

The present invention also provides a gene encoding the protein.

The present invention also provides a microarray for diagnosing a liver related disease comprising an antibody to the gene or the protein.

The present invention also provides a primer set for diagnosing liver related diseases consisting of primers for the gene.

The present invention also provides a kit for diagnosing a liver related disease comprising the primer set.

The present invention also provides a method for diagnosing liver disease of a mammal using the primer set.

By providing the fact that the MML2 gene is specifically expressed in liver tissue, the inventors can easily access information such as protein-protein networks, pathways or transcriptional regulation of genes, and the fact that the MML2 gene is a liver specific gene. This can reduce false positives in diagnostic studies of liver-related diseases. In addition, the MML2 gene can be used as a marker for the diagnosis of liver-related diseases, making it easier to diagnose.When designing a drug as a therapeutic agent for disease symptoms, the drug target is reduced to a protein for MML2. This allows for faster treatment at an early stage.

In order to achieve the above object, the present invention provides an MML2 protein that is specifically expressed in liver tissue, consisting of the amino acid sequence represented by SEQ ID NO: 2.

Prediction of tissue-specific genes through methods such as DNA chips does not make it possible to distinguish whether they are actually tissue-specific genes or experimental errors due to the numerous errors present in the experiment.

However, if the candidate group is identified as a tissue-specific gene by performing an Audic-Claverie Test based on the EST (Expressed Sequence Tag) and using experiments such as RT-PCR, the candidate group can be judged as a genetic influence factor. Reliability is increased. The inventors have provided higher reliability for candidate groups of tissue specific genes by examining candidate groups of genes involved in tissue development and examining whether they contain tissue specific information.

Thus, the present invention provides MML2 protein that is specifically expressed in liver tissue. The range of MML2 proteins according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably 80%, more preferably 90%, and even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution or deletion of amino acids Preferably it refers to a protein having a sequence homology of 95% or more, and exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2.

The MML2 protein according to the invention is preferably derived from, but not limited to, a mouse.

The MML2 protein according to the present invention can be used as a target for therapeutic drugs for liver related diseases. Examples of liver-related diseases include, but are not limited to, hepatitis, liver cancer, fatty liver, cirrhosis, and the like. When designing drugs as diagnostics for liver-related disease symptoms, drug targets can be reduced with MML2 protein, enabling faster diagnosis at the onset of the onset.

The present invention also provides a gene encoding the MML2 protein. Genes of the invention include both genomic DNA and cDNA encoding the MML2 protein. Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene comprises a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 can do. The "% sequence homology" for a polynucleotide is determined by comparing the comparison regions with two optimally arranged sequences, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The invention also provides an antibody against the MML2 protein according to the invention. The antibody of the present invention can be produced by a conventional method from a protein obtained by cloning a gene into an expression vector according to a conventional method to obtain a protein encoded by the MML2 gene. This includes partial peptides that can be made from the protein, and the partial peptide of the present invention includes at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibody of this invention also contains special antibodies, such as a humanized antibody.

The present invention also provides a marker for diagnosing liver related diseases, comprising the MML2 gene according to the present invention. MML2 gene of the present invention may preferably have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of liver-related diseases include, but are not limited to, hepatitis, liver cancer, fatty liver, cirrhosis, and the like. By measuring the severity of liver-related diseases according to the expression level of the gene, it is possible to establish a correlation between the expression level of the gene and the severity of the disease. Based on the established correlations, only the expression level of the gene can be measured to predict and diagnose liver-related diseases of the subject. Since the gene can be used as a marker for detecting or diagnosing a genetic tendency of a liver-related disease, it is possible to diagnose and perform treatment before the disease occurs by using such information.

The invention also provides a microarray for diagnosing liver related diseases, comprising the MML2 gene according to the invention or an antibody against the MML2 protein of the invention. MML2 gene of the present invention may preferably have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of liver-related diseases include, but are not limited to, hepatitis, liver cancer, fatty liver, cirrhosis, and the like. By measuring the severity of liver-related diseases according to the expression level of the gene, it is possible to establish a correlation between the expression level of the gene and the severity of the disease. Based on the established correlations, only the expression level of the gene can be measured to predict and diagnose liver-related diseases of the subject. The microarray of the present invention can be easily produced by a manufacturing method commonly used in the art.

In the microarray of the present invention, "microarray" means that a polynucleotide or an antibody group is immobilized at a high density on a substrate, and the polynucleotide or antibody group means a microarray each immobilized in a predetermined region. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The term “substrate” refers to any substrate to which a polynucleotide or antibody probe can be coupled under conditions that retain hybridization properties and keep the background level of hybridization low. Typically, the substrate may be a microtiter plate, membrane (eg, nylon or nitrocellulose) or microspheres (beads) or chips. Prior to application or immobilization on the membrane, nucleic acid probes can be modified to promote immobilization or to improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH 2 groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

The invention also provides a primer set for diagnosing liver related diseases consisting of sense and antisense primers of the MML2 protein coding gene of the invention. The sense and antisense primers may be preferably composed of the nucleotide sequence of SEQ ID NO: 3 and 4, respectively. Examples of liver-related diseases include, but are not limited to, hepatitis, liver cancer, fatty liver, cirrhosis, and the like. After separating RNA from the tissue, RT-PCR was performed using the primer set to measure the severity of liver-related diseases according to the amount of RT-PCR products to be amplified. Correlation of severity of Based on the established correlations, only the presence and quantity of RT-PCR products can be measured to predict and diagnose liver related diseases in the subject.

In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

As used herein, oligonucleotides used as primers may also include nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

The present invention also provides a primer set according to the present invention; And kits for diagnosing liver related diseases, including reagents required for RT-PCR. In the kit of the present invention, the reagents required for the RT-PCR may be, for example, but not limited to, a forward primer and a reverse primer, a reverse transcriptase, a ribonuclease inhibitor, a heat resistant polymerase, a reaction buffer, and the like, and a reaction buffer Silver includes both reverse transcriptase and PCR buffer, and heat resistant polymerase may be EF Taq polymerase and the like, and reverse transcriptase is a reverse transcriptase originating from various sources, for example, avian myeloblastosis virus-derived reverse transcriptase. (avian myeloblastosis virus-derived virus reverse transcriptase (AMV RTase)), mouse leukemia virus-derived virus reverse transcriptase (MuLV RTase) and Rouse-associated virus 2 reverse transcriptase 2 reverse transcriptase (RAV-2 RTase).

The kit may also include instructions. The guide is a printed document that explains how to use the kit, such as how to prepare reverse transcription buffer and PCR buffer, and the reaction conditions presented. The instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

The present invention also provides

Isolating total RNA from the mammalian sample;

Amplifying a target sequence by using the isolated whole RNA as a template and performing RT-PCR using a primer set according to the present invention; And

It provides a method for diagnosing liver disease in a mammal, comprising detecting the amplification product.

After separating RNA from the tissue, RT-PCR was performed using the primer set to measure the severity of liver-related diseases according to the amount of RT-PCR products to be amplified. Correlation of severity of Based on the established correlations, only the presence and quantity of RT-PCR products can be measured to predict and diagnose liver related diseases in the subject. Examples of liver-related diseases include, but are not limited to, hepatitis, liver cancer, fatty liver, cirrhosis, and the like.

As a method for separating total RNA from a mammalian sample of the present invention, a method known in the art may be used, for example, a phenol extraction method may be used. Using the isolated whole RNA as a template, the target sequence can be amplified by performing RT-PCR using a primer set according to an embodiment of the present invention as a primer. Such PCR methods are well known in the art, and commercially available kits may be used.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. The oligonucleotide primer set used to amplify the target sequence is as described above.

The method of the present invention includes detecting the amplification product. Detection of the amplification product may be performed through DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis can be performed. Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling substance, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, radioactivity measuring method is to add a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash Radioactivity can be measured using a liquid scintillation counter.

In the method of the present invention, the mammal is preferably a human, but may also include non-human animals including pets such as dogs and cats, cattle such as cattle, sheep, pigs, horses, and experimental animals such as rats, white papers, guinea pigs, and the like. have.

The present invention also provides a method of diagnosing liver related disease in a mammal comprising measuring the expression of the MML2 gene according to the present invention. MML2 gene of the present invention may preferably have a nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto. Examples of liver-related diseases include, but are not limited to, hepatitis, liver cancer, fatty liver, cirrhosis, and the like. By measuring the severity of liver-related diseases according to the expression level of the gene, it is possible to establish a correlation between the expression level of the gene and the severity of the disease. Based on the established correlations, only the expression level of the gene can be measured to diagnose liver-related diseases of the subject. The mammal is preferably a human, but may also include non-human animals including pets such as dogs or cats, livestock such as cattle, sheep, pigs, horses, and experimental animals such as rats, white papers, guinea pigs, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the invention, so the scope of the invention is not to be construed as limited by these examples.

Example

Example 1 Establishment of Liver Specific Gene Candidate Group Using TISA Database

Noh SJ 2006 [DNA Res 13 (5) 229-43] published a TISA (Tissue-specific Alternative Splicing) database containing information on genes, transcript structure, alternative splicing events and tissue specificity. TISA is a database that identifies tissue-specific genes by clustering EST data containing information on the expressed tissue into a genetic map and verifying that the number of ESTs of the corresponding TISA genes is statistically significant. If there was a gene predicted as a liver tissue specific gene in TISA, it was extracted as a candidate group of liver tissue specific gene.

Example 2: RT-PCR Experiment of Liver Specific Gene Candidates

Total RNA was used as mouse total RNA of Biochain. There are 15 different types of RNA tissue used, including muscle, stomach, testis, thymus, heart, cerebellum, cerebral and liver. ), Colon, kidney, lung, spleen, ovary, pancreas and uterus. Roche Co. CDNA was synthesized according to the Ltd RT-PCR kit manual and PCR was performed. The primers used were as follows, all designed through eprimer3 primer design software and in-house python wrapping script. PCR conditions were finally extended for 72 min 10 min after 35 cycles of pre-denatured 94 ° C. 3 min, followed by 94 ° C. 30 sec, annealing 58-60 ° C. 30 sec, extension 72 ° C. 1 min. 1% agarose gel electrophoresis was performed to confirm the PCR product.

Primer sequence:

Forward primer: 5'-CCACTCCGAATTCCCATGA-3 '(SEQ ID NO: 3)

Reverse primer: 5'-GAAGCAGGAAGGGCCTGTTA-3 '(SEQ ID NO: 4)

Figure 2 shows the RT-PCR results of the MML2 gene. As can be seen in Figure 2, it was found that the MML2 gene is rarely expressed in other tissues, but specifically expressed in liver tissue.

Example 3: Reference of Liver Specific Gene Candidates with UniProt

UniProt (http://www.ebi.uniprot.org) was developed in 2002 as an EBI / SIB Swiss-Prot, TrEMBL database PIR-Protein Sequence Database (PIR-PSD). It is a way to combine the contents into a UniProt consortium and provide them with mutual cooperation. The consortium provides comprehensive, accurate information about protein sequences, and maintains an accessible interface despite providing many references.

In order to identify which protein candidates appear to be specific to the liver, and which functions are linked to which proteins on UniProt, the sequence data of the liver specific gene candidates was applied to the BLAST alignment provided by UniProt's website. Only data with values greater than or equal to% are taken.

BLAST results showed 99% (423/424) identity with Serpin A11 in mice. Serpin A11 belongs to the Serpin superfamily, most of which are known to play various roles through the inhibition of serine protease. In addition to inhibitory functions, functions such as hormone transport and tumor suppression have been reported. They form a specific tertiary structure in various organs and inhibit protease activity. Serpin A11 has not yet been identified as a function related to diseases such as hypertension, cirrhosis, dementia and cancer.

1 illustrates a system for analyzing liver specific genes.

Figure 2 shows the RT-PCR results of the MML2 gene.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Liver specific gene MML2 <130> PN08203 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1445 <212> DNA <213> Mouse <400> 1 gcacagccaa attcagattt attagaactg aagcacaggg gtcctgccat gcaggggggt 60 cctgccgtgc aggttgggcc ttcctgtggc ctccccacca caattaccca gcagctgggt 120 tgaccacttt ccccaagaag agcagactct gggtggtcac ctcccagagc agaagcagga 180 agggcctgtt atagtgggcc tgtggggctg atgtcatgtt cagggctggg ggctgggaca 240 ggaggccaga ggcagctgca gcctcagttc ccttctcgtt catgtccacg atggccttgt 300 gtgacaccct ggagacagtt ttgttgagtt gccccataat tcctgataag tcagcttcca 360 tgtcgaatag gttgccaaga ccaatgaggg gcaggatctc ttccaggttg taagttgcgg 420 aaattgaaaa ccttggcagg tgcaaatcca acagactggg caggaaccgc tggccccacc 480 ttctcagcgt ctctggctgg agggcagcct ctacctgctg catcttccca gggtcaggga 540 ggaccagcag cagcaaggcg gtgccactgt actcgatctg gaggacagtg cacgacgcct 600 cctggtcgta caggaacctg tgcatttcct tctgtcgcat catgggaatt cggagtggcg 660 tcctctggtc cagggagaag ctctcctgct tccgggtctg gtagcgatca aagggatgct 720 tgcacttggc tttgaagaag atgtagttca agagaaccat gagagtgtca tggctgaact 780 ccgggagaca gcccaccacc tgcccgtagg tctgcttcct cactaggtca ttgatctgct 840 gccctgtggc agctgcttct gtgaagttgg cagaaaaggc cagagctcca tacagttcct 900 tggcactgtc cagaaaacgt tgctgaggct tcagctgtct gtctaggaac aggaaatgtc 960 ccagtttcag ttccagtttg gggctgggga ggtcaagagt gtgcaggagg ctctggaaac 1020 cccggtggac gtcagctgct ggggtctctg tgaggttgaa gcccaggctc tccagaattt 1080 gagtctgggt gtcagcatgt gccccaagag acagcagagc caggctgctg gagaggctca 1140 caggggagaa aaggatgttt cccgctactt cctctgctag ttgcttgtat agacgcaaag 1200 caaaattcgt aatggtgggt gtgaccttgt gataggcggg tgctggctcc agggactggt 1260 gactggctgg ttgagatgcc cccagactct tatctccatg ggcagaaaat ggctgacaat 1320 ggacaggaag caggacctcg gctaccagca gccacagcca cagccacact ggccccatca 1380 tagagaagtc acccaggtgg gcccagctcc acagtcccgt aatgcctcct gcccacaggg 1440 tgata 1445 <210> 2 <211> 424 <212> PRT <213> Mouse <400> 2 Met Gly Pro Val Trp Leu Trp Leu Trp Leu Leu Val Ala Glu Val Leu   1 5 10 15 Leu Pro Val His Cys Gln Pro Phe Ser Ala His Gly Asp Lys Ser Leu              20 25 30 Gly Ala Ser Gln Pro Ala Ser His Gln Ser Leu Glu Pro Ala Pro Ala          35 40 45 Tyr His Lys Val Thr Pro Thr Ile Thr Asn Phe Ala Leu Arg Leu Tyr      50 55 60 Lys Gln Leu Ala Glu Glu Val Ala Gly Asn Ile Leu Phe Ser Pro Val  65 70 75 80 Ser Leu Ser Ser Ser Leu Ala Leu Leu Ser Leu Gly Ala His Ala Asp                  85 90 95 Thr Gln Thr Gln Ile Leu Glu Ser Leu Gly Phe Asn Leu Thr Glu Thr             100 105 110 Pro Ala Ala Asp Val His Arg Gly Phe Gln Ser Leu Leu His Thr Leu         115 120 125 Asp Leu Pro Ser Pro Lys Leu Glu Leu Lys Leu Gly His Phe Leu Phe     130 135 140 Leu Asp Arg Gln Leu Lys Pro Gln Gln Arg Phe Leu Asp Ser Ala Lys 145 150 155 160 Glu Leu Tyr Gly Ala Leu Ala Phe Ser Ala Asn Phe Thr Glu Ala Ala                 165 170 175 Ala Thr Gly Gln Gln Ile Asn Asp Leu Val Arg Lys Gln Thr Tyr Gly             180 185 190 Gln Val Val Gly Cys Leu Pro Glu Phe Ser His Asp Thr Leu Met Val         195 200 205 Leu Leu Asn Tyr Ile Phe Phe Lys Ala Lys Cys Lys His Pro Phe Asp     210 215 220 Arg Tyr Gln Thr Arg Lys Gln Glu Ser Phe Ser Leu Asp Gln Arg Thr 225 230 235 240 Pro Leu Arg Ile Pro Met Met Arg Gln Lys Glu Met His Arg Phe Leu                 245 250 255 Tyr Asp Gln Glu Ala Ser Cys Thr Val Leu Gln Ile Glu Tyr Ser Gly             260 265 270 Thr Ala Leu Leu Leu Leu Val Leu Pro Asp Pro Gly Lys Met Gln Gln         275 280 285 Val Glu Ala Ala Leu Gln Pro Glu Thr Leu Arg Arg Trp Gly Gln Arg     290 295 300 Phe Leu Pro Ser Leu Leu Asp Leu His Leu Pro Arg Phe Ser Ile Ser 305 310 315 320 Ala Thr Tyr Asn Leu Glu Glu Ile Leu Pro Leu Ile Gly Leu Gly Asn                 325 330 335 Leu Phe Asp Met Glu Ala Asp Leu Ser Gly Ile Met Gly Gln Leu Asn             340 345 350 Lys Thr Val Ser Arg Val Ser His Lys Ala Ile Val Asp Met Asn Glu         355 360 365 Lys Gly Thr Glu Ala Ala Ala Ala Ser Gly Leu Leu Ser Gln Pro Pro     370 375 380 Ala Leu Asn Met Thr Ser Ala Pro Gln Ala His Tyr Asn Arg Pro Phe 385 390 395 400 Leu Leu Leu Leu Trp Glu Val Thr Thr Gln Ser Leu Leu Phe Leu Gly                 405 410 415 Lys Val Val Asn Pro Ala Ala Gly             420 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 ccactccgaa ttcccatga 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 4 gaagcaggaa gggcctgtta 20  

Claims (9)

delete delete A microorganism for diagnosing a liver-related disease selected from the group consisting of hepatitis, liver cancer, fatty liver and cirrhosis, comprising a gene encoding the MML2 protein specifically expressed in liver tissue consisting of the amino acid sequence represented by SEQ ID NO: 2 Array. The microarray of claim 3, wherein the gene is formed of a nucleotide sequence represented by SEQ ID NO: 1. Microarray for diagnosing liver related diseases selected from the group consisting of hepatitis, liver cancer, fatty liver and cirrhosis, comprising an antibody against MML2 protein specifically expressed in liver tissue consisting of the amino acid sequence represented by SEQ ID NO: 2 . A primer set for diagnosing liver related diseases selected from the group consisting of hepatitis, liver cancer, fatty liver and cirrhosis, consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4. A primer set according to claim 6; And a hepatitis, liver cancer, fatty liver and cirrhosis, comprising a reagent required for RT-PCR. Separating total RNA from non-human animal samples; Amplifying a target sequence by using the isolated whole RNA as a template and performing RT-PCR using the primer set according to claim 6; And Detecting the amplification product, the liver related disease selected from the group consisting of hepatitis, liver cancer, fatty liver and cirrhosis of non-human animals. delete

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