KR20120060790A - Bio-markers for detecting tissue-specific expression in bovine's muscle regions, primer sets for detecting the bio-markers and methods of identification of bovines' muscle regions using the same - Google Patents

Abstract

PURPOSE: A bovine muscle-specific biomarker and a primer are provided to be used for genetic identification in a cow. CONSTITUTION: A bovine muscle-specific biomarker contains MYL2 gene with base of sequence number 5. A primer set for detecting the biomarker contains a primer pair having a base of sequence number 15 and a base of sequence number 16. A method for determining a shank part comprises: a step of isolating and culturing muscle stem cell from a test sample; a step of differentiating muscle cells or adipocytes; a step of isolating RNA from the stem cells, muscle cells, or adipocytes; a step of preparing cDNA using the isolated RNA; a step of performing real-time PCR; and a step of analyzing the PCR result.

Description

A set of primers for detecting specific biomarkers specific to the cow's muscle area and specific biomarkers of cow's muscle area, and how to use the same {BIO-MARKERS FOR DETECTING TISSUE-SPECIFIC EXPRESSION IN BOVINE'S MUSCLE REGIONS, PRIMER SETS FOR DETECTING THE BIO-MARKERS AND METHODS OF IDENTIFICATION OF BOVINES' MUSCLE REGIONS USING THE SAME}

The present invention relates to a set of primers for detecting a specific biomarker for a bovine muscle region, a biomarker specific for a bovine muscle region, and a method of using the same. More specifically, it relates to a cow muscle region-specific biomarker containing a specific gene for a cattle avalanche region, a primer set for detecting a cattle muscle region-specific biomarker, and a method of discriminating a cattle avalanche region using the same.

The meat industry has been regarded as an internationally important industry from the past to the present, and the importance of the meat industry is increasingly being emphasized in Korea as the amount of meat consumption increases with increasing income and changes in dietary habits (meat consumption per capita in 2003 was about 33.3 kg). ).

In particular, beef is an important high-quality protein source that provides various flavors in various parts, and shows a considerable price difference depending on the amount and taste produced by each part. For example, Yangji is only produced at about 10% per head from slaughtered cattle, showing about twice the price difference when comparing the price with cheap parts (e.g., embossed meat, beef tongue, and tongue). Therefore, it is common to sell cheap beef parts as if they are expensive parts.

This is because it is very difficult to classify each part of meat as part of the bone, and to solve this problem, the Ministry of Food, Agriculture, Forestry and Fisheries revised the classification method for each part of meat by grade and by type. Looking at the matters related to the division and sale of meat by part, it is stipulated that when the division of meat is sold by part, the name of the largely divided part is used to separate and sell by part. However, when the divided parts are sold together, there is no regulation on the labeling of the relevant section. It was made possible by setting labeling regulations that the mixed sale of parts was not possible (FY2008). However, despite such amendments to the law, the sales of deceived parts continue to prevail, and these events are occurring more and more due to the import of cheap imported beef resulting from the conclusion of the FTA.

Recently, efforts are being made to solve the above problems by using gene identification techniques in each variety, individual, and partial meat state, but studies on the discovery of specific genes according to regions are still insufficient. Therefore, there is an urgent need to develop a biomarker, such as a gene specific to a muscle region in cattle.

An object of the present invention is to provide a biomarker specific to a muscle region of cattle.

Another object of the present invention is to provide a primer set for detecting a biomarker specific to a muscle region of the cow.

Another object of the present invention is to provide a method for discriminating a cow's avalanche area using a primer set for detecting the cow's muscle site-specific biomarker or the cow's muscle site-specific biomarker.

In order to achieve the above object, the present invention comprises a thymidylate synthase, tropoelastin (Tropoelastin, elastin precursor), RDM1, CV980409.1-A:r, MYL2 or thioredoxin-acting protein (TXNIP) gene It provides a biomarker specific to the muscle region of the cow.

The inventors of the present invention extracted and cultured muscle stem cells from four muscle areas of cattle, loin, yangji, and red bean to differentiate into muscle cells and differentiate into adipocytes (heterodifferentiation), and then three types of cells (muscle After extracting each RNA from stem cells, cells differentiated into muscle, and cells differentiated into fat), the level of gene expression is observed using a DNA chip containing about 20,000 cow-related genes (cattle, loin, yangji, red bean paste). At each site, muscle stem cells + cells differentiated into muscle, muscle stem cells + cells differentiated into fat were analyzed). Based on the results of the DNA chip, 138 muscle region-specific genes were selected (eg, muscle stem cells and cells differentiated into muscle cells), such that they are expressed more than 4 times in one area and less than 2 times in the remaining 3 areas (e.g., muscle stem cells and cells differentiated into muscle cells). Genes that are expressed more than 4 times in cells differentiated into muscle in a situation by comparing and analyzing gene expressions in, and whose expression is less than twice as much in loin, Yangji, and Red Beans, or genes whose analysis values of DNA chips differ by more than 2 times compared to other sites. Selection) was analyzed using each cell sample used for DNA chip analysis using a real-time PCR method. The 30 genes identified in this way were analyzed by real-time PCR method (cell stage, tissue stage) for actual condition, loin, yangji, and red bean musculature, and compared with the other three sites, 6 genes that were highly expressed in the situation (thymid delay). T. synthase, Tropoelastin (elastin precursor), RDM1, CV980409.1-A:r, MYL2 or thioredoxin-acting protein (TXNIP) gene) were found. In other words, it was confirmed that the gene can be used as a biomarker specific to the muscle region of cattle.

The biomarker may be a gene specifically expressed in the avalanche muscle region of the cow's muscle region.

The level of expression of the biomarker in any one of muscle stem cells, cells differentiated into muscle, or cells differentiated into adipose in a muscle region may be specific for cells of the same type in other muscle regions of cattle.

The thymidylate synthase gene may be specific for muscle stem cells, which are cells of the same type in other muscle regions of cattle, in which the level of expression of the muscle stem cells is specific. That is, the thymidylate synthase gene may have a greater degree of expression of muscle stem cells at the site of the avalanche than that of muscle stem cells at other sites.

The tropoelastin (elastin precursor), RDM1, CV980409.1-A:r, or MYL2 gene expresses the level of expression of the cells differentiated into the muscle relative to the cells differentiated into muscle, which are cells of the same type in other muscle regions of cattle. It can be specific. That is, the tropoelastin (elastin precursor), RDM1, CV980409.1-A:r, or MYL2 gene may have a greater degree of expression of cells differentiated into muscles at the site of the avalanche than cells differentiated into muscles at other sites. .

The thioredoxin-acting protein gene may be specific for a cell differentiated into adipose, which is a cell of the same type in another muscle region of a cow, in which the degree of expression of the cell differentiated into adipose. That is, the thioredoxin-acting protein gene may have a higher level of expression of cells differentiated into fat at the avalanche site than cells differentiated into fat at other sites.

In order to achieve the above other object, the present invention provides a primer set for detecting a specific biomarker for a muscle region of cattle. The bovine muscle-specific biomarkers include thymidylate synthase, tropoelastin (elastin precursor), RDM1, CV980409.1-A:r, MYL2 or thioredoxin-acting protein (TXNIP) gene. The set of primers for detecting a specific biomarker for the bovine muscle region includes SEQ ID NOs: 7 to 18. However, the primer sequence is not limited in size and position to bind to the template as long as it can amplify the nucleotide sequence of the biomarker or some nucleotide sequence in the biomarker, and those skilled in the art can easily use the primer selection software. The design is possible.

A primer set consisting of a pair of the nucleotide sequence of SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 8 may be used to detect the thymidylate synthase gene.

A primer set consisting of a pair of a nucleotide sequence of SEQ ID NO: 9 and a nucleotide sequence of SEQ ID NO: 10 may be used to detect a tropoelastin (elastin precursor) gene.

A primer set consisting of a pair of the nucleotide sequence of SEQ ID NO: 11 and the nucleotide sequence of SEQ ID NO: 12 can be used to detect the RDM1 gene.

A primer set consisting of a pair of the nucleotide sequence of SEQ ID NO: 13 and the nucleotide sequence of SEQ ID NO: 14 can be used to detect the CV980409.1-A:r gene.

A primer set consisting of a pair of the nucleotide sequence of SEQ ID NO: 15 and the nucleotide sequence of SEQ ID NO: 16 can be used to detect the MYL2 gene.

A primer set consisting of a pair of a nucleotide sequence of SEQ ID NO: 17 and a nucleotide sequence of SEQ ID NO: 18 can be used to detect a thioredoxin-acting protein (TXNIP) gene.

In order to achieve the above still another object, the present invention provides a method of determining the avalanche area of a cow using a biomarker specific to the muscle area of the cow. The bovine muscle region-specific biomarkers include thymidylate synthase, tropoelastin (elastin precursor), RDM1, CV980409.1-A:r, MYL2 or thioredoxin-acting protein (TXNIP) gene. The bovine muscle region-specific biomarker includes a gene specifically expressed in the avalanche muscle region.

It is possible to determine whether the cow's muscle tissue site is an avalanche by using real-time-PCR analysis of the cow's muscle tissue site-specific biomarker. At this time, for real-time-PCR analysis, muscle stem cells can be extracted and cultured for each muscle part of cattle (sad, sirloin, yangji, red bean curd). By adjusting the culture conditions, it is possible to differentiate into muscle stem cells and stem cells into muscle cells and into adipocytes (heterodifferentiation). After extracting total RNA (total RNA) from muscle stem cells, cells differentiated into muscle, and cells differentiated into fat, when synthesizing cDNA, only mRNA is synthesized as cDNA using oligo(d)T primer. Han-PCR analysis can be performed using the cDNA product and each gene-specific primer. The gene-specific primer may include SEQ ID NO: 7 to SEQ ID NO: 18, which is a primer set for detecting a biomarker specific for a region of the bovine muscle tissue.

The method for determining the avalanche area of a cow according to the present invention comprises the steps of isolating and culturing muscle stem cells from a specimen; Differentiating the muscle stem cells into muscle cells or adipocytes; Extracting RNA from the muscle stem cells, muscle cells or adipocytes; Obtaining cDNA using the extracted RNA; One or two or more selected from the group consisting of the cDNA, thymidylate synthase, tropoelastin (Tropoelastin, elastin precursor), RDM1, CV980409.1-A:r, MYL2 and thioredoxin-acting protein (TXNIP) Performing real-time-PCR using a biomarker-specific primer set specific to a bovine muscle region including the gene; And analyzing the real-time-PCR result.

According to the present invention, only muscle stem cells are isolated from a sample sample, that is, each muscle area of a cow, and differentiated into muscle stem cells, muscle cells, or adipocytes to analyze the expression pattern of a specific biomarker of a cow's muscle area expressed in the cells. As a result, it can be determined whether the specimen is a situation. Accordingly, by comparing and determining the expression of specific genes in the muscle region of the cow, a method capable of detecting whether the muscle region of the cow is an avalanche with high reliability is provided.

The biomarker specific to the muscle region of the cow obtained according to the present invention can be used as a useful marker for the identification of the gene for each region of the cattle related to the classification of each region of the cattle. In addition, the primer set for detecting a specific biomarker for a muscle region of the cow may be effectively used for detection of a biomarker specific for a muscle tissue of the cow.

1 is a flow chart showing a gene analysis process used as a specific biomarker for a muscle region of a cow according to the present invention,
2A to 2C are cells used in the present invention, respectively, showing muscle stem cells, cells differentiated into muscle, and cells differentiated into fat,
3A to 3F are graphs showing the results of analysis in cells using a real-time PCR analysis method (cell stage) based on the DNA chip result,
4A to 4F are graphs showing the results obtained using a real-time PCR analysis method (tissue step) of the gene identified in the cell step in an actual muscle region.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited by the examples.

<Example 1> Muscle stem cell extraction, cell culture and differentiation

1. Muscle stem cell extraction, cell culture and differentiation

Muscle stem cell extraction is a conventional procedure as described in Ethanol Inhibits Skeletal Muscle Cell Proliferation and Delays Its Differentiation in Cell Culture, author: Garriga J et al., Alcohol and Alcoholism Vol. 35, pp 236-241, 2000). It was carried out according to. After rinsing the muscles of the cattle slaughtered on the same day with physiological saline, chop the muscles of the slaughtered slaughter, sirloin, yangji, and hongdukkae on the same day with scissors and tweezers. USA) was added and digested for 2 hours in a shaking thermostat at 37° C. at 90 rpm so that digestion could be efficiently carried out throughout the tissues. The digested tissue was centrifuged at about 90 g for about 3 minutes, and the supernatant was recovered using a 40 μm strainer, followed by centrifugation at about 2,500 rpm for 20 minutes. After centrifugation was completed, the supernatant was discarded and the washing medium was added. After centrifugation at 2,500 rpm for 5 minutes, the supernatant was discarded and centrifugation was repeated once more under the same conditions to remove the supernatant. Cells were suspended in culture medium (DMEM (HyClone Laboratories, Logan, UT, USA) + 10% FBS (fetal bovine serum, HyClone) + penicillin/streptomycin (10,000 units/ml, Invitrogen, Carlsbad, CA, USA)). Then, the isolated muscle stem cells are cultured at 37°C. In the case of stem cells, culture was performed in DMEM + 10% FBS + penicillin/streptomycin for about 8 days (see Fig. 2-A), For the differentiation of muscle stem cells (see Fig. 2-A) into muscle cells (see Fig. 2-B), culture was performed in DMEM + 10% FBS + penicillin / streptomycin for 14 to 21 days, and differentiation into adipocytes. For (see Fig. 2-C), the differentiation medium (see Table 1) was cultured for 7 days after treatment. Table 1 shows the composition of the trans-differentiation-inducing medium.

0.1 M ascorbic acid 200 uM 33 mM Biotic (1000 x) 33 uM 34 mM Pantothenate (2000 x) 17 uM Caprylic acid 1 mM 2 N acetic acid 10 mM 10 mg/ml insulin 10 ug/ml 5 mM dexamethasone (Dexmathasone,) 5 uM 3-Isobuthyl-1-methylxanthine 0.5 mM Thiazolidione (PPAR? ligand) 10 uM

< Example 2> mRNA Extraction and DNA Chip analysis

1.Total RNA extraction

Total RNA (Total RNA) was extracted after culturing the cells extracted from each of Satae, Loin, Yangji, and Red Beans into muscle stem cells, cells differentiated into muscle, and cells differentiated into fat. At this time, in the case of muscle stem cells, total RNA (Total RNA) was extracted without fusion between cells. In the case of cells differentiated into muscle, total RNA (Total RNA) was extracted in the presence of several nuclei through fusion between cells. That is, the cells differentiated into muscle were extracted with total RNA (Total RNA) after the formation of a myotube. The medium was removed, about 1 ml of Trizol solution was added per 100 mm culture dish, collected in a 1.5 ml tube, and the cell membrane was crushed using a grinder. About 200 µl of chloroform was added thereto, left at room temperature for about 10 minutes, centrifuged at about 12,000 rpm for about 10 minutes, and then the supernatant was transferred to a new tube. About 500 µl of isopropanol was added to the tube to which the supernatant was transferred, and allowed to stand at room temperature for about 10 minutes, followed by centrifugation to obtain a total RNA (Pellet). For washing, about 70% ethanol was added, centrifuged at about 7,500 rpm for about 5 minutes, discarded the supernatant, dried total RNA, and then added diethylpyrocarbonate (DEPC)-treated water. Put it in. At this time, if DEPC-treated water is added according to the total RNA (Total RNA) pellet size, it is easy to finally adjust the total RNA (Total RNA) amount to a similar concentration even if the total RNA (Total RNA) pellet size is different. .

2. DNA chip analysis

Using a DNA chip (Texas A&M, USA) designed by Christine Elscik's team of Tesxas A&M, where approximately 20,000 genes are integrated, gene expression between cells differentiated into muscle and cells differentiated into adipose based on stem cells is used. The degree was observed. At this time, in order to compare the gene expression levels of cells differentiated into muscle or fat for each site, the sample combinations for each tissue DNA chip analysis were prepared in all eight combinations.

Sample combination for DNA chip analysis

1) Situation: Stem cells + cells differentiated into muscle (1), Stem cells + cells differentiated into fat (2)

2) Loin: Stem cells + cells differentiated into muscle (3), Stem cells + cells differentiated into fat (4)

3) Yangji: cells differentiated into stem cells + muscle (5), cells differentiated into stem cells + fat (6)

4) Red bean: stem cells + cells differentiated into muscle (7), stem cells + cells differentiated into fat (8)

The DNA chip analysis was performed by comparing RNAs extracted from muscle stem cells and cells differentiated into muscle, and muscle stem cells and cells differentiated into adipose for each muscle region (repeated 3 each). Each 1 ㎍ of total RNA was left for 10 minutes at 65 ℃ with T7 primer and then cDNA master mix (5X First strand buffer, 0.1 M dithiothreitol (DTT), 10 mM dNTP mix, RNase-Out, and Molony Murine Leukemia Virus-reverse transcriptase; MMLV-RT) and left at 40° C. for 2 hours and 65° C. for 15 minutes to synthesize dsDNA. The transcription of dsDNA was performed with a transcription master mix (4X Transcription buffer, 0.1 M DTT, NTP mix, 50% polyethylene glycol, RNase-Out, Inorganic pyrophosphatase, T7-RNA polymerase, and Cyanine 3/5-CTP) and 40 ℃. It proceeded for 2 hours at. Amplified and probe hybridized (Cy 3 for stem cells, Cy5 for muscle differentiated cells, and fat differentiated cells) cRNA was purified by cRNA Cleanup Module (Agilent Technologies), and the concentration and purity of the purified cRNA Was measured using an ND-1000 spectrophotometer (NanoDrop Technologies). To check the efficiency of the probe, cRNA was added to 80 µl hybridization reagent (5X SSC, 0.1% sodium dodecyl sulfate (SDS), and 30% formamide) and left at 100° C. for 2-3 minutes. After standing, it was added directly to the DNA chip using a pipette and left in a humid hybridization chamber (GenomicTree, Seoul, Korea) for about 42°C for 16 hours. The hybridized DNA chip was washed twice for 5 minutes in 2X SSC/0.1% SDS, 10 minutes in 0.1X SSC/0.1% SDS, and 2 minutes in 0.1X SSC, and then dried immediately using a chip centrifuge ( Genomic Tree, Daejeon, Korea).

4. Results

When comparing muscle stem cells and cells differentiated into muscle or cells differentiated into adipose in the avalanche muscle region, gene or DNA chip analysis values that are expressed more than 4 times and less than 2 times in the rest are compared with other sites 2 Genes that differed more than two times were observed. In the case of the six genes shown in Table 2 below, the gene expression pattern was specifically shown only in the avalanche site, and the other three sites (sirloin, yangji, hongdukkae) showed low specificity in the gene expression pattern. For each of the six genes, the level of gene expression of cells differentiated into muscle or fat relative to muscle stem cells was compared with respect to each site, and this was shown by the DNA chip analysis values in Table 2 below. Here, the DNA chip analysis value is a numerical value of the expression ratio of different types of cells based on the expression of one gene for one type of cell during DNA chip analysis. At this time, in the analysis shown in Table 2, in the case of muscle stem cells> cells differentiated into muscle, it is quantified based on the expression amount of cells differentiated into muscle (the value of cells differentiated into muscle is set to 1 and the remaining values are calculated). In the case of muscle stem cells <muscle differentiated cells and muscle stem cells <fat differentiated cells, the expression amount of muscle stem cells (muscular stem cells value is set to 1 and the remaining values are calculated) were quantified. Of the six genes, one gene (Thymidlyate synthase) highly expressed in muscle stem cells in a state of change, four in cells differentiated into muscle (Tropoelastin, Elastin precursor), RDM1 (Similar to RAD25 motif-containing protein 1), CV980409.1-A:r and MYL2 (Myosin light 2, regulatory cardiac, slow)), 1 in adipose-differentiated cells (Thioredoxin-interacting protein , TXNIP)). Table 2 shows the DNA chip results.

analysis gene GeneBank accession ID DNA Chip analysis value ( fold ) situation sirloin Sunny Red bean Muscle stem cells>cells differentiated into muscle Thymidlyate synthetase NM_001037816.1 3.86 0.26 0.32 0.27 Muscle stem cells <cells differentiated into muscle Tropoelastin (elastin precursor) NM_175772 3.90 1.27 0.67 1.07 RDM1 (Similar to RAD25 motif-containing protein 1) XM_591173 6.89 0.62 1.99 1.03 CV980409.1-A:r CV980409 4.32 0.79 1.37 0.93 MYL2 (Myosin light 2, regulatory cardiac, slow) NM_001035025.1 10.53 0.88 0.83 3.77 Muscle stem cells <cells differentiated into fat Thioredoxin-interacting protein (TXNIP) NM_001101875 7.56 1.84 0.89 1.38

< Example 3> Real-time ( Real - time ) PCR Analysis (cell level)

1. Real-time PCR analysis

Based on the results obtained through the DNA chip analysis, when comparing muscle stem cells and cells differentiated into muscle or cells differentiated into adipose in one tissue site, genes that are expressed more than 4 times and less than 2 times in the remaining sites, or After selecting a gene (see Table 2) whose DNA chip analysis value differs by more than two times compared to other sites, a primer was designed (see Table 3). The primers were designed to amplify some nucleotide sequence portions of the entire sequences of the genes, and the nucleotide sequence numbers and sizes in the genes are as shown in Table 3. At this time, the nucleotide sequence information registered in the National Center for Biotechnology Information was used, and the primer was designed with Primer 3 software (http://frodo.wi.mit.edu). Thereafter, the concentration of the extracted RNA sample was measured using an ND-100 spectrometer (NanoDrop Technologies, Wilmington, DE, USA). The measured RNA was reverse transcribed into single-stranded cDNA using Superscript-II reverse transcriptase (Invitrogen). Total RNA (1 μg in a total volume of 20 μL) was raised _{and started with d(T) 20} primers (meaning that 20 T bases are attached, Bioneer, Daejeon, Korea), and reverse transcription was initiated at 42° C. for 50 minutes, and It was carried out at 72° C. for 15 minutes. Then, PCR was performed using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using 2 µl of the 10X diluted cDNA product and 10p moles (10 pmoles) of each gene-specific primer (see Table 3). Performed. SYBR Green (Power SYBR Green PCR Master Mix, Applied Biosystems, USA) was used as a fluorescent source. Real-time PCR was performed under the following conditions: pre-denaturation of the synthesized cDNA for 10 minutes at 95°C, denaturation at 95°C for 33 seconds, annealing at each gene-specific primer Tm (°C) and 33 at 72°C. It was carried out with 40 cycles of extension for seconds. Proper amplification of the gene of interest was verified by melting point analysis and 1.2% agarose gel electrophoresis. The primers shown in Table 3 below were used. Table 3 shows the primer sequences used for gene analysis.

Put up ID GeneBank accession ID size ( bp ) Base sequence Forward direction primer Reverse primer Thymidylate synthetase NM_001037816.1 194 Base sequence 1 5'-ctacgtggtgaatggggagt-3' (base sequence 7) 5'-tcagtggctcgatgtgattc-3'
(Base sequence 8) Tropoelastin
(Elastin precursor) NM_175772 204 Base sequence 2 5'-caaaccaggacaaggatgga-3' (base sequence 9) 5'-tccagttggtacccaagcat-3' (base sequence 10) Similar to RAD52 motif-containing protein 1 XM_591173 207 Base sequence 3 5'-caggctttcttcctgcaaac-3' (base sequence 11) 5'-tgtaacctgggacccttgag-3' (base sequence 12) CV980409.1-A:r CV980409 201 Base sequence 4 5'-tcaggtgcttcacacaaatg-3' (base sequence 13) 5'-ccactacaccctcaccctgt-3' (base sequence 14) Myosin, light　 chain 2, regulatory, cardiac, slow (MYL2) NM_001035025.1 205 Base sequence 5 5'-gcgtgtgaacgtgaaaaatg-3' (base sequence 15) 5'-cgttgtcagcatctccttga-3' (base sequence 16) Thioredoxin-interacting protein (TXNIP) NM_001101875 191 Base sequence 6 5'-aagcacaatgacagggaaaaa-3' (base sequence 17) 5'-gcaacatctggatgctctca-3' (base sequence 18)

2. Results

3A to 3F are graphs showing results of analysis in cells using a real-time PCR analysis method (cell stage) based on the DNA chip result. The graph shows the degree of expression (fold difference) of genes expressed in each muscle cell.

As shown in FIGS. 3A to 3F, it was confirmed that the six genes were specifically expressed in the avalanche site compared to the rest of the regions (sirloin, yangji, hongdukkae). In addition, it was confirmed that one of the six genes was highly expressed in muscle stem cells in a state of change, four in cells differentiated into muscle, and one in cells differentiated into adipose.

DNA Based on the chip result, real-time PCR Genes identified by analysis

1) Genes highly expressed in the state of the art muscle stem cells: thymidylate synthase (see Fig. 3a)

2) Genes highly expressed in cells differentiated into the state of the art muscle: Tropoelastin (elastin precursor) (see FIG. 3B), RDM1 (see FIG. 3C), CV980409.1-A:r (see FIG. 3D) and MYL2 (See Fig. 3e)

3) Gene that is highly expressed in cells differentiated into adipose of a state: thioredoxin-acting protein (TXNIP) (see Fig. 3f)

<Example 4> Real-time PCR analysis (tissue step)

1. Experimental method

Total RNA was extracted from the six genes mentioned in Example 3 from actual tissues (sad, sirloin, yangji, red bean curd). Total RNA (Total RNA) extraction was carried out by putting 1 ml of trizol (Traizol reagent, Invitrogen) into about 0.2 g of each tissue and pulverizing with a pulverizer (homogenizer). About 200 µl of chloroform was added thereto, left at room temperature for about 10 minutes, centrifuged at about 12,000 rpm for about 10 minutes, and then the supernatant was transferred to a new tube. About 500 µl of isopropanol was added to the tube to which the supernatant was transferred, and allowed to stand at room temperature for about 10 minutes, followed by centrifugation to obtain a total RNA (Pellet). For washing, about 70% ethanol was added, and after centrifugation at about 7,500 rpm for about 5 minutes, the supernatant was discarded, total RNA was dried, and DEPC-treated water was added. At this time, if DEPC-treated water is added according to the total RNA (Total RNA) pellet size, it is easy to finally adjust the total RNA (Total RNA) amount to a similar concentration even if the total RNA (Total RNA) pellet size is different. . The extracted RNA was dissolved in DEPC-treated water and stored at -80°C until analysis. The concentration of RNA was measured using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The measured RNA was reverse transcribed into single-stranded cDNA using Superscript-II reverse transcriptase (Invitrogen). Total RNA (1 μg in a total volume of 20 μL) was raised (dT) with ₂₀ primers (meaning that 20 T bases are attached, Bioneer, Daejeon, Korea), and reverse transcription was initiated at 42° C. for 50 minutes, and 72 It was carried out at °C for 15 minutes. After that, PCR was performed using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) using 2 µl of the 10X diluted cDNA product and 10p moles (10pmoles) of each gene-specific primer (see Table 3). I did. SYBR Green (Power SYBR Green PCR Master Mix, Applied Biosystems, USA) was used as a fluorescent source. Real-time PCR was performed under the following conditions: pre-denaturation of the synthesized cDNA for 10 minutes at 95°C, denaturation at 95°C for 33 seconds, annealing at each gene-specific primer Tm (°C) and 33 at 72°C. It was carried out with 40 cycles of extension for seconds. Proper amplification of the gene of interest was verified by melting point analysis and 1.2% agarose gel electrophoresis.

2. Experiment result

4A to 4F are graphs showing the results obtained using a real-time PCR analysis method (tissue step) of the gene identified in the cell step in an actual muscle region. That is, FIGS. 4A to 4F are graphs showing the level of expression in each muscle tissue region of each of the six selected genes. In FIGS. 4A to 4F, the result of calculating the gene expression value in the rest of the site with the value of the gene expressed in the avalanche tissue as 100 is shown. As a result of the above experiment, it was confirmed that all of the six genes were highly expressed in the situation compared to the loin, yangji, and hongdukkae regions. In addition, as shown in Figs. 3a to 3b, it was confirmed that one of the six genes is highly expressed in muscle stem cells of the situation, four in cells differentiated into muscle, and one in cells differentiated into adipose. .

<110> Industry-Academic Cooperation Foundation, Yeungnam University <120> BIO-MARKERS FOR DETECTING TISSUE-SPECIFIC EXPRESSION IN BOVINE'S MUSCLE REGIONS, PRIMER SETS FOR DETECTING THE BIO-MARKERS AND METHODS OF IDENTIFICATION OF BOVINES' MUSCLE REGIONS USING THE SAME <130> DP-2010-0079 <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 194 <212> DNA <213> Bos taurus <400> 1 ctacgtggtg aatggggagt tgtcctgcca gttgtaccag cggtcggggg acatgggcct 60 gggtgtgccc ttcaacattg ccagctacgc cctgctcacc tacatgatcg cacacatcac 120 ggacctgaag ccaggtgact tcgtgcacac tctgggagat gcacacattt acctgaatca 180 catcgagcca ctga 194 <210> 2 <211> 204 <212> DNA <213> Bos taurus <400> 2 caaaccagga caaggatgga cggacggatg gacagatggg cggatgaatt ccacaagagc 60 aagagggctg ttcccaatat gggggcgggg ggagtcttcg gacataggga acagcaatgg 120 gtcggggaaa ggggggccaa ggaggccaag agatggtgtg acccaatcca gcgtgagggt 180 ttcgcatgtt gggtaccaac tgga 204 <210> 3 <211> 207 <212> DNA <213> Bos taurus <400> 3 caggctttct tcctgcaaac ttatcatatg caaaagttta ggtgatttgc atcatcttct 60 ggcccggagg cctttttctc taaaggtgat tattctaaag tcaggcgcca gcctccaaaa 120 agcattaaat aaagttgcat tcctacagag caaaggtgtg gtgggctata acaagaaaaa 180 gaattaactc aagggtccca ggttaca 207 <210> 4 <211> 201 <212> DNA <213> Bos taurus <400> 4 tcaggtgctt cacacaaatg tatacagagg tcttaggggt gttacatcat cttctggcca 60 ggggggcctg ctgacaattt acgaccctct ccttgtgaca gcggtcagtc aatcaggaca 120 cttatttctc cagggctgat tattctcaaa acagacgcca cccaaataaa gttacattcc 180 tacagggtga gggtgtagtg g 201 <210> 5 <211> 205 <212> DNA <213> Bos taurus <400> 5 gcgtgtgaac gtgaaaaatg aggaaattga tgaaatgctc aaggaggctc caggtcccat 60 taactttact gtgttcctac agatgttcgg ggagaaactt aaaggagcag atcccgagga 120 gaccattctc aacgcgttca aagtgtttga ccctgaaggc aaaggagtgc tcaaggctga 180 ttatatcaag gagatgctga caacg 205 <210> 6 <211> 191 <212> DNA <213> Bos taurus <400> 6 aagcacaatg acagggaaaa aaaagtgtta ctgcttttga gactttgtcg cagtgtacgg 60 aattctgcag ttctgacact gattacgtaa gggttgctgc tatcagcctt gcccactgtg 120 acttctccaa acccaaggag gaaccctaga taaaatgccc caacactgtg atgagagcat 180 ccagatgttg c 191 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for thymidylate synthetase <400> 7 ctacgtggtg aatggggagt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for thymidylate synthetase <400> 8 tcagtggctc gatgtgattc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for tropoelastin <400> 9 caaaccagga caaggatgga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for tropoelastin <400> 10 tccagttggt acccaagcat 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for RDM1 <400> 11 caggctttct tcctgcaaac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for RDM1 <400> 12 tgtaacctgg gacccttgag 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for CV980409.1-A:r <400> 13 tcaggtgctt cacacaaatg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for CV980409.1-A:r <400> 14 ccactacacc ctcaccctgt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MYL2 <400> 15 gcgtgtgaac gtgaaaaatg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MYL2 <400> 16 cgttgtcagc atctccttga 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TXNIP <400> 17 aagcacaatg acagggaaaa a 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TXNIP <400> 18 gcaacatctg gatgctctca 20

Claims (10)

Biomarker specific for the muscle region of a cow comprising the MYL2 gene. The biomarker according to claim 1, wherein the muscle part of the cow is a landslide site. The bovine muscle site-specific biomarker according to claim 2, wherein the expression level of the cells differentiated into muscles at the avalanche site is the MYL2 gene larger than the cells differentiated into muscles from other sites. The method of claim 3, wherein the MYL2 gene comprises a nucleotide sequence of 5 bovine muscle region-specific biomarker. A primer set for detecting a muscle region specific biomarker of a cow of claim 1. The primer set according to claim 5, wherein the primer set consists of a pair of nucleotide sequences of SEQ ID NO: 15 and a nucleotide sequence of SEQ ID NO: 16, and is capable of detecting MYL2 gene. A method of determining the avalanche site of a cow using a muscle-specific biomarker of a cow containing the MYL2 gene. The method of claim 7, wherein the bovine muscle region specific biomarker is used for real-time PCR analysis. The method of claim 8, wherein the real-time PCR analysis uses a set of primers for detecting a biomarker specific to a muscle region of a cow. Separating and culturing muscle stem cells from a sample;
Differentiating the muscle stem cells into muscle cells or adipocytes;
Extracting RNA from the muscle stem cells, muscle cells or adipocytes;
Obtaining cDNA using the extracted RNA;
Performing real-time PCR using the cDNA and a bovine muscle region specific biomarker specific primer set comprising a MYL2 gene; And
Analyzing the real-time PCR results.

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