CN114814221A - Thyroid cancer early diagnosis marker and application thereof - Google Patents
Thyroid cancer early diagnosis marker and application thereof Download PDFInfo
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Abstract
The invention discloses an application of 14-3-3 gamma in preparing a thyroid cancer diagnosis product, which utilizes the high specificity expression of 14-3-3 gamma protein in thyroid cancer tissues and takes the 14-3-3 gamma protein as a serum marker for diagnosing thyroid cancer, so that the thyroid cancer diagnosis method has the comprehensive advantages of earlier diagnosis time, simple and rapid operation, no wound, low cost and the like.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a thyroid cancer early diagnosis marker and application thereof.
Background
Thyroid nodules are clinically very common, are the tumors with the highest rate of incidence increase in recent 30 years, account for 5% of the global population, and 7-15% of them are thyroid cancers. Asymptomatic benign thyroid nodules can be followed up regularly, while malignant nodules need surgical treatment, however, in recent years, the clinical divergence of low-risk suspicious nodules is large, and therefore, the identification of benign and malignant thyroid nodules becomes a key basis for guiding clinical treatment.
The ultrasound-guided fine needle puncture cytology examination (FNA) is the most common examination method for identifying the benign and malignant thyroid nodules at present, has the advantages of being minimally invasive, easy to operate and the like, and is the gold standard for identifying the benign and malignant thyroid nodules at present. However, this method is an invasive examination, and the technique is not yet widespread nationwide due to the limitations of the apparatus and the varying level of puncture technique for each doctor. At present, the sensitivity of FNA is 83 percent (65 to 98 percent), the specificity is 92 percent (72 to 100 percent), the positive predictive value is 75 percent (50 to 96 percent), the false negative rate is 5 percent (1 to 11 percent), and the false positive rate is 5 percent (0 to 7 percent). The reasons for the false negative of FNA are analyzed by reference to domestic and foreign literature summaries as follows: the technical reasons for needle aspiration and puncture and smear making include that the nodules are too small and deep, or the nodules are large and only part of the nodules are cancerated, so that effective cell components are not collected; the nodules are not clearly demarcated from the surrounding tissue, the tumor range cannot be determined, and the puncture may not really penetrate into the nodules; less tissue is sucked, or too much blood causes too little cell mass; smear film making quality is poor. The diagnosis of thyroid malignant tumors of certain histopathology types by FNAC is difficult. Such as follicular papillary carcinoma, medullary thyroid carcinoma, and proliferative nodules remain difficult to identify. And thirdly, diagnosis standards are difficult to master, and misdiagnosis and missed diagnosis are easy to cause. Therefore, a more sensitive and non-invasive diagnostic method for identifying benign and malignant thyroid nodules is urgently needed, and the invention provides a novel early thyroid cancer marker.
The invention discloses a transcriptome sequencing method, which is a feasible method for discovering functional genes and markers.
Disclosure of Invention
One of the purposes of the present invention is to provide a serum marker related to thyroid cancer.
Another object of the present invention is to provide a reagent for detecting the above-mentioned serum marker.
The invention also aims to provide a diagnostic kit for thyroid cancer.
The fourth purpose of the invention is to provide the application of the serum marker in preparing a thyroid cancer diagnostic reagent: obtaining a blood sample of a subject to be detected, and detecting 14-3-3 gamma protein in the serum sample by an Elisa method, wherein the protein level of a YWHAG gene expression product 14-3-3 gamma protein in the blood of a papillary thyroid cancer patient is obviously increased; detecting the expression level of 14-3-3 gamma in the thyroid cancer cell strain by a western-blot method after the human thyroid cancer cell is cultured; the expression and cell localization of 14-3-3 gamma in thyroid cancer and tissues beside the cancer are detected by immunofluorescence in the obtained tissue sample of the object to be detected, the tissue sample of the object to be detected is obtained, and the expression difference of 14-3-3 gamma in thyroid cancer and tissues beside the cancer is detected by qPCR.
The fifth purpose of the invention is to provide the application of the serum marker and the detection reagent of the serum marker in the preparation of a thyroid cancer diagnosis kit, and the kit for diagnosing thyroid cancer comprises a reagent for specifically detecting serum 14-3-3 gamma protein.
The inventor discovers that 14-3-3 gamma protein is related to thyroid cancer by separating and researching 14-3-3 gamma protein in serum of thyroid cancer patients and healthy people, and the 14-3-3 gamma protein determined by large sample screening has high specificity expression in cells, tissues and peripheral blood of thyroid cancer patients, and the expression level of 14-3-3 gamma in thyroid cancer is higher than that of healthy controls or benign thyroid nodules, so that the thyroid cancer diagnostic kit has a good diagnostic value for thyroid cancer.
The CDS region sequence of human YWHAG gene is queried at NCBI and 14-3-3 gamma is transcribed, wherein the protein sequence of 14-3-3 gamma is queried at the website https:// www.ncbi.nlm.nih.gov/protein/BAA85184.1, and the sequence of YWHAG gene is queried at the website https:// www.ncbi.nlm.nih.gov/nuccore/NM _ 012479.4.
The invention has the advantages that:
the expression level of the 14-3-3 gamma protein in cells, tissues and peripheral blood (serum) provided by the invention can identify the benign and malignant thyroid nodules at an early stage, and the method for diagnosing the thyroid cancer by using the marker has the sensitivity and specificity of 98.2 percent and 96.6 percent respectively in some embodiments;
the methods of the invention provide for thyroid cancer identification, or risk, by "diagnosing" or "providing a thyroid cancer diagnosis," generally meaning providing a reference for making clinical decisions;
the 14-3-3 gamma protein is specifically and highly expressed in the serum of a thyroid cancer patient, and the application of the 14-3-3 gamma protein relates to a thyroid cancer serum marker and a thyroid cancer noninvasive diagnosis kit or method;
the 14-3-3 gamma protein is highly expressed in cells, tissues and peripheral blood (serum) of thyroid cancer, can further improve the diagnosis accuracy of FNA, and is used as an auxiliary diagnosis method of FNA.
Drawings
FIG. 1: comparing the concentration of 14-3-3 gamma protein in serum of papillary thyroid carcinoma patients with that of normal human serum;
FIG. 2: ROC plot of diagnostic value of 14-3-3 gamma protein;
FIG. 3: western Blot is used for detecting the expression level of 14-3-3 gamma protein in thyroid cancer cells and normal thyroid cells;
FIG. 4: detecting the relative expression level and cell sub-localization of 14-3-3 gamma protein in thyroid cancer tissues and tissues beside the thyroid cancer by an immunofluorescence method;
FIG. 5: and qPCR is used for detecting the relative expression level of YWHAG gene in thyroid cancer cells and normal thyroid cells.
Detailed Description
The present application is further illustrated with reference to specific examples. The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
Example 1: and (3) verifying the value of 14-3-3 gamma serving as a serum diagnosis marker of the thyroid cancer patient by using ELISA.
Subject inclusion and exclusion criteria:
source of case
All cases were from hospitalized patients in Hunan ya Hospital, southern university, 12 months between 2017 and 2019. The blood samples of the normal control group are all from healthy physical examination persons in Hunan ya Hospital, southern China university, and are diseases without heart, brain, lung, liver and kidney diseases and cancer diseases and diseases without known influence on research indexes.
Diagnostic criteria
Thyroid cancer diagnosis was based on histopathological examination and staging was based on AJCC TNM staging system (eighth edition). Firstly, receiving a papillary thyroid cancer excision operation; ② the pathological diagnosis after operation is papillary thyroid carcinoma; thirdly, the follow-up visit data is complete; fourthly, preoperative serum and postoperative tissue specimens exist; all specimens come from the same hospital case exclusion standard: hashimoto's thyroiditis; ② benign thyroid nodule; ③ malignant tumors; fourthly, the clinical data are incomplete; infectious diseases; sixthly, do not wish to participate in the study.
Healthy controls were included as standard: thyroid color ultrasonography shows healthy physical examination people without thyroid nodules, and physical examination is normal; ② the indexes of blood, urine, stool, blood sedimentation, liver function, kidney function, electrolyte, blood sugar and blood fat are normal; and thirdly, no other malignant tumors or metabolic diseases exist.
Preparation of standard substance
1. Preparation of 14-3-3 gamma protein
(1) Constructing a recombinant prokaryotic expression plasmid of papillary thyroid carcinoma-associated protein 14-3-3 gamma by using a gene cloning technology;
(2) respectively transforming the constructed recombinant prokaryotic expression plasmids into escherichia coli B121(DE3), and inducing the expression of the target protein by IPTG (isopropyl thio-hemistanin);
(3) according to the label carried by the target protein, the target protein is purified by adopting a traditional corresponding purification scheme;
(4) protein concentration was determined by Bradford assay and the immunological activity of the purified protein was characterized by Western Blot
2. Antigen coating solution: carbonate buffer, pH 9.6;
3. sealing liquid: PBST buffer containing 2% (W/V) BSA;
4. enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein;
5.96-well ELISA plate: purchased from Abcam corporation;
6. color developing solution A: 0.02% (W/V) TMB, preparation: dissolving 0.005g of methylbenzidine (TMB) in 25ml of deionized water;
7. color developing solution B: 0.006% (W/V) carbamide peroxide, formulation: 4.665g of citric acid and 4.40g of Na2HPO418.40g of citric acid are fully dissolved in 400ml of deionized water, 3.2ml of 0.75% carbamide peroxide is added, the pH value is adjusted to 5.0-5.5, deionized water is added to the mixture until the volume is up to 500ml, and the mixture is uniformly mixed and stored at 4 ℃;
8. positive control serum: live 14-3-3 gamma antibody positive human serum;
9. negative control serum: inactivated 14-3-3 gamma antibody negative human serum;
10.14-3-3 gamma antibody standard.
Thirdly, preparing an antigen-coated ELISA plate
1. Preparing papillary thyroid carcinoma-associated antigen solution: dissolving 14-3-3 gamma protein in the antigen coating solution, and mixing well to prepare antigen solution, wherein the concentration of 14-3-3 gamma protein is 0.75 ug/ml;
2. coating an enzyme label plate: adding the prepared 14-3-3 gamma antigen solution into sample application holes of a 96-hole enzyme label plate, wherein the sample application amount is 100 ul/hole, 3 positive control holes and 4 negative control holes are arranged, and 1 blank control hole is reserved. Coating 14-3-3 gamma antigen solution in 3 positive control holes and 4 negative control holes, only adding coating solution in 1 blank control hole, and removing the coating solution after overnight at 4 ℃;
3. and (3) sealing: adding a sealing solution into the coated sample application holes of the 96-hole enzyme label plate, incubating for 2 hours at room temperature, and removing the redundant sealing solution;
4. drying the treated 96-well enzyme label plate in a drying box at 37 ℃, packaging, and storing at 4 ℃ for later use
Composition of ELISA kit
(1) 96-hole enzyme label plate coated by antigen;
(2)14-3-3 gamma antibody standard;
(3) positive control serum: inactivated 14-3-3 gamma antibody positive human serum;
(4) negative control serum: inactivated 14-3-3 gamma antibody negative human serum;
(5) sample diluent: PBST buffer containing 1% (W/V) BSA;
(6) enzyme-labeled secondary antibody: horse radish peroxidase-labeled RecA protein, purchased from Abcam;
(7) secondary antibody dilution: PBST buffer containing 1% (W/V) BSA;
(8) color development liquid: the color development liquid consists of a color development liquid A and a color development liquid B. Wherein. The color developing solution A is 0.02% (W/V) TMB, and the color developing solution B is 0.006% (W/V) carbamide peroxide;
(9) stopping liquid: 10% concentrated sulfuric acid;
(10) washing liquid: PBST (phosphate) buffer 0.01M, pH 7.2.
Elisa to verify the expression of 14-3-3 gamma protein
(1) Preparing reagents in an ELISA kit, collecting 68 healthy controls and 166 thyroid cancer patients, extracting serum, and obtaining 100ul serum samples;
(2) and adding 100ul of diluted positive control serum into the positive control hole, adding 100ul of diluted negative control serum into the negative control hole, taking the rest reaction holes coated with the 14-3-3 gamma antigen as sample holes to be detected, and adding 100ul of sample to be detected into each hole. The blank control hole is not added with the sample, and the operation of the rest steps is the same as that of the sample hole to be detected. Preparing 5 standard solutions with different concentrations by using the sample diluent and the 14-3-3 gamma antibody standard, simultaneously arranging a group of 5 standard holes, and respectively adding 100ul of a series of standard solutions with different concentrations into the standard holes for drawing a standard curve. Incubating for 2 hours at 37 ℃ after the sample addition is finished;
(3) removing the redundant liquid in the reaction hole, and washing for 5 times by using a washing solution;
(4) adding 0.1ml of freshly diluted enzyme-labeled secondary antibody into each reaction hole, and incubating at 37 ℃ for 1 hour;
(5) removing the redundant liquid in the reaction hole, and washing for 5 times by using a washing solution;
(6) mixing the color development liquid A and the color development liquid B in an equal volume of 1:1, adding 100ul of the mixed color development liquid into each reaction hole, and reacting for 30 minutes at 37 ℃;
(7) adding 90ul of stop solution into each reaction hole, stopping color development, and reading an absorbance value A at a wavelength of 450nm within 5 minutes;
(8) and (4) judging a result: connecting the values of the concentrations point by taking the concentration of the standard substance as an abscissa and the absorbance as an ordinate to obtain an S-shaped standard curve for quantitative determination;
(9) the threshold for serum 14-3-3 gamma antibody levels was set at 0pg/ml and any serum with a 14-3-3 gamma antibody concentration greater than this was considered positive.
Sixth, statistical method
Experiments were completed in 3 replicates per sample, the results were the average of 3 replicates, statistical analysis was performed using SPSS22.0 statistical software, and non-parametric rank-sum tests were used between different groups, considering that P <0.05 is statistically significant.
Seventh, experimental results
1. As shown in fig. 1, the level of the protein of the ywcag gene expression product 14-3-3 γ in blood of papillary thyroid cancer patients is significantly increased compared to the normal group, and the difference is statistically significant (P < 0.001);
2. from the serum ELISA results of 166 patients with pathological section diagnosed as papillary thyroid carcinoma and 88 healthy persons, ROC graphs were obtained, and the total ability of the kit to distinguish between patients and healthy persons was evaluated by finding the optimal cutoff value from the upper limit. The result is shown in FIG. 2, where the area under the ROC curve is 0.873 and the optimum cutoff value is 2.33.
According to the cut-off value obtained by the ROC curve, the esophageal cancer can be diagnosed by calculating the expression level of 14-3-3 gamma in serum detected by an ELISA method, the sensitivity is up to 98.2 percent, and the specificity is 96.6 percent.
Example 2: the expression level of 14-3-3 gamma in the thyroid cancer cell strain is detected by using a western-blot method.
The experimental method comprises the following steps:
1. and (3) cell culture, namely culturing an immortalized human normal thyroid cell line (Nthy), culturing human thyroid cancer cells (TPC-1, BCPAP, K1 and KTC-1) and RPMI1640 complete culture medium containing 10% FBS, putting the cells in a CO2 humid incubator for culture at 37 ℃, and detecting by using a mycoplasma detection kit to eliminate mycoplasma pollution after culture.
2. Protein extraction and immunoblotting (Western Blot, WB) to detect protein expression
(1) Protein extraction: adherent cells are collected by trypsinization or cell scraping, washed with precooled PBS 3 times, centrifuged, and then the PBS is aspirated, and 90-120ul of protein extraction reagent (RIPA and protease inhibitor 100:1) is added. Shaking at high speed for 5 seconds, incubating on ice for 20 minutes, shaking at high speed for 5 seconds, centrifuging at 4 ℃ (12000rpm,15 minutes), and transferring the supernatant to another clean, pre-cooled EP tube. Placed on ice for subsequent experiments or stored at-80 ℃.
(2) Protein concentration determination: (a) and (3) carrying out equal-fold dilution on standard protein with known concentration, determining the absorbance of the standard protein with different concentration, thereby drawing a protein concentration standard curve, substituting the absorbance value of the protein sample with unknown concentration into a standard curve regression equation, and obtaining the concentration of the unknown protein sample. (b) Removing appropriate amount of protein from each group of samples, diluting with lysate to obtain equal-concentration equal-volume sample to be loaded, adding 1/5 volumes of 5X protein loading buffer solution, mixing, denaturing at 100 deg.C for 5min, and storing at-80 deg.C for subsequent WB experiment or cryopreservation.
(3) Immunoblotting experiments
(a) The formulation of SDS-PAGE gels (5% acrylamides) is shown in Table 1 below:
TABLE 1
(b) The SDS-PAGE gels were formulated as follows:
TABLE 2
(c) Protein loading and electrophoresis: taking equivalent protein samples after heating denaturation, grouping and adding samples according to preset, wherein the uplifting amount of each pore protein is 20-60ug, carrying out electrophoresis in a standard electrophoresis buffer solution, and carrying out electrophoresis conditions: and (4) keeping the voltage at 80V, increasing the voltage to 120V after the sample enters a separation gel Marker strip for clear separation, stopping electrophoresis when the bromophenol blue dye reaches the bottom of the gel or the protein Marker reaches a target position, taking the SDS-PAGE gel down, putting the SDS-PAGE gel into a membrane transfer buffer solution, and preparing to perform a next membrane transfer experiment.
(d) Protein membrane conversion: the protein transfer membrane is divided into a semi-dry transfer membrane and a wet transfer membrane, the laboratory mainly adopts a wet transfer membrane method, and the transfer membrane is arranged in a sandwich way: the method comprises the steps of placing a film-rotating sandwich into a 1X standard film-rotating solution, wherein the film-rotating conditions comprise constant current of 300mA on ice, film-rotating time of 70-100 minutes, determining the film-rotating time according to the target molecular weight, and distinguishing the positive electrode and the negative electrode of the film-rotating.
(e) And (3) sealing of the membrane: after the membrane conversion is finished, the PVDF membrane is put into skimmed milk powder or BSA (bovine serum albumin) prepared by 5% TBST (TBST) for sealing, is sealed for 45min-2h at normal temperature, is washed for 5 seconds by TBST, and then enters the next step of antibody incubation.
(f) Incubating the primary antibody: diluting 14-3-3 gamma antibody and GAPDH antibody with an anti-diluent (1:2000), cutting a PVDF membrane strip containing a target gene according to a protein marker, putting the PVDF membrane strip into the antibody diluent to ensure that the PVDF membrane is completely covered by the antibody, and incubating overnight (generally 12h-16h) on a shaking table at 4 ℃.
(g) Incubation of secondary antibody: primary antibody was incubated overnight, washed 3 times with TBST for 8min each time, and the residual primary antibody was removed. And (3) diluting the secondary antibody by using a secondary antibody diluent (1:5000), putting the PVDF membrane strip into the secondary antibody diluent to ensure that the PVDF is completely covered by the antibody and has no air bubbles in the middle, and shaking and incubating at room temperature for 45min-2 h.
(h) Color development: after the secondary antibody incubation is completed, washing for 8min for 3 times by TBST, removing residual secondary antibody, and mixing ECL luminescence solution A and solution B in a ratio of 1:1 proportion to prepare working solution, putting the PVDF membrane protein surface incubated by the antibody on a preservative film, uniformly dropwise adding a proper amount of ECL luminous working solution, putting the strip into a gel scanning system and an electrophoresis image analysis processing system for scanning and developing, and storing a strip scanning picture, or exposing the strip by using an X-ray film and scanning and storing.
3. Results of the experiment
As shown in fig. 3, normal thyroid cells: nthy
Thyroid cancer cell: KTC-1, K1, BCPAP, TPC-1
Compared with a normal thyroid cell strain, the expression level of 14-3-3 gamma in a thyroid cancer cell strain is obviously increased, relative quantitative statistical analysis is carried out, and the difference between the two is of statistical significance.
Example 3: immunofluorescence detects the expression and cell localization of 14-3-3 gamma in thyroid cancer and tissues beside the cancer.
Subject inclusion and exclusion criteria:
source of case
All cases were from hospitalized patients in Hunan ya Hospital, southern university, 12 months between 2017 and 2019.
Diagnostic criteria
Thyroid cancer diagnosis was based on histopathological examination and staging was based on AJCC TNM staging system (eighth edition). Firstly, receiving a papillary thyroid cancer excision operation; ② the pathological diagnosis after operation is papillary thyroid carcinoma; thirdly, the follow-up visit data is complete; fourthly, corresponding thyroid cancer and tissue specimens beside the cancer exist; all specimens come from the same hospital. Case exclusion criteria: hashimoto's thyroiditis; ② benign thyroid nodule; ③ malignant tumors; fourthly, the clinical data are incomplete; infectious diseases; sixthly, do not wish to participate in the study.
Second, concrete operation method
1. Freezing and slicing, fixing with 4% paraformaldehyde or 95% ethanol for 15-30 min, and air drying at room temperature for 15 min;
soaking in PBS for 10min, and repeating for 3 times;
3.0.5% Tritonx-100 is transparent for 20 minutes at room temperature;
4, washing with PBS for 10 minutes, and repeating for 3 times;
5. blocking with 10% normal goat serum or BSA at room temperature for 1 hour;
6. adding primary antibody, and incubating overnight in a wet box at 4 ℃ in the dark;
PBS washing for 10min, repeating for 3 times;
8. adding a fluorescence-labeled secondary antibody (1:200), and incubating for 1 hour at 37 ℃ in a wet box in the absence of light;
PBS washing for 10min, repeating for 3 times;
10. sheep serum diluted DAPI (1:1000) was incubated for 10min at 37 ℃;
PBS washing for 10min, repeating for 3 times;
12. sealing the fluorescence-quenching agent, and observing under a fluorescence microscope
Third, experimental results
See fig. 4, expression of the ywtag gene in thyroid cancer tissues and paracarcinoma tissues. Wherein, red fluorescence signals are observed in the thyroid cancer tissues, and no red fluorescence signals exist in the tissues beside the cancer, so that the relative expression level of YWHAG genes in the thyroid cancer tissues is obviously higher than that of the tissues beside the cancer. Further statistical analysis showed that the differences were statistically significant.
Example 4: qPCR detection of expression difference of YWHAG gene in normal thyroid cell and thyroid cancer cell
The experimental method comprises the following steps:
1. and (3) cell culture, namely culturing an immortalized human normal thyroid cell line (Nthy), culturing human thyroid cancer cells (TPC-1, BCPAP, K1 and KTC-1) and RPMI1640 complete culture medium containing 10% FBS, putting the cells in a CO2 humid incubator for culture at 37 ℃, and detecting by using a mycoplasma detection kit to eliminate mycoplasma pollution after culture.
2. Extraction of RNA from cells
(1) Adding 1ml of Trizol into a T25 cell culture bottle, fully and uniformly mixing, transferring into an enzyme-free centrifuge tube, and standing for 5min at room temperature;
(2) adding 200 μ l chloroform into the centrifuge tube, reversing, mixing, and standing at room temperature for 10 min;
(3) setting the rotating speed of a centrifugal machine to be 12000rpm, and putting the centrifugal machine into a centrifugal tube for centrifugation for 10 min;
(4) dividing the liquid into three layers, transferring the supernatant of the upper layer into a new enzyme-free centrifuge tube, adding precooled isopropanol with the same volume, mixing uniformly, and standing on ice for 10 min;
(5) setting the rotating speed of a centrifugal machine to be 12000rpm, and putting the centrifugal machine into a centrifugal tube for centrifugation for 10 min;
(6) discarding the supernatant, adding 1ml of 75% ethanol prepared from DEPC water into the precipitate, mixing, placing in a centrifuge, and centrifuging at 7500rpm for 10 min;
(7) removing supernatant, placing in an ultraclean workbench, drying RNA white precipitate, adding 50 μ l DEPC water to dissolve precipitate, and detecting RNA purity and concentration with a trace quantitative detector.
Reverse transcription of cDNA
The operation was carried out according to TAKARA reverse transcription kit
(1) Removal of genomic DNA
A10. mu.l reaction system was prepared: 5 Xg DNA Eraser Buffer 2.0. mu.l, gDNA Eraser 1.0. mu.l, TotalRNA 1.0. mu.g, RNase Free dH2O up to 10. mu.l.
Reaction conditions of the PCR apparatus: 42 ℃ for 2 minutes, 4 ℃.
(2) Reverse transcription reaction
Prepare 20. mu.l reaction system: 10. mu.l of the reaction solution of step (1), 1.0. mu.l of PrimeScript RT Enzyme Mix I, 1.0. mu.l of RT Primer Mix, 24.0. mu.l of 5 XPimeScript Buffer, and 4.0. mu.l of RNase FreedH 2O 4.0.
Reaction conditions of the PCR apparatus: 15 minutes at 37 ℃, 5 seconds at 85 ℃ and 4 ℃.
4. Fluorescent quantitative PCR reaction
(1) Primers were designed based on the sequence ENST00000307630.5 of the YWHAG gene, the sequence being as follows:
a forward primer: 5'-AACAGACGCAGGGGTAAGAAT-3'
Reverse primer: 5'-CTGCTCAATGCTACTGATGACC-3'
With GAPDH as an internal reference, the primer sequences of GAPDH are as follows:
forward primer 5'-GGAGCGAGATCCCTCCAAAAT-3'
Reverse primer 5'-GGCTGTTGTCATACTTCTCATGG-3'
(2) Reaction reagents are prepared according to a TAKARA fluorescent quantitative PCR kit:
reaction reagents, SYBR Green Premix Ex Taq (2X) 10. mu.l, forward primer 0.4. mu.l, reverse primer 0.4. mu.l, cDNA template 2. mu.l, ddH2O 7.2.2. mu.l were prepared.
The parameters of the fluorescent quantitative PCR instrument are set as follows: 5min at 95 ℃; 38 cycles of 95 ℃ for 15s and 60 ℃ for 40s
(3) The relative expression level of YWHAG was calculated by the 2-DELTA. Ct method using GAPDH as a reference gene.
Third, experimental results
The difference in the expression level of YWHAG gene between normal thyroid cells and thyroid cancer cells is shown in FIG. 5. It can be seen that the relative expression level of YWHAG gene in thyroid cancer cell is obviously higher than that of normal thyroid cell, and the statistical analysis shows that the difference has statistical significance.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (4)
1. A diagnostic marker for thyroid cancer, comprising: the diagnostic marker is 14-3-3 gamma protein.
2. The diagnostic marker of claim 1, wherein: the diagnostic marker is a serum marker.
3. The diagnostic marker of claim 2, wherein the serum marker is used for preparing a reagent for diagnosing and detecting thyroid cancer.
4. The diagnostic marker of claim 2, wherein the serum marker and the detection reagent for the serum marker are used for preparing a thyroid cancer diagnostic kit, and the kit comprises a reagent for specifically detecting serum 14-3-3 gamma protein.
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