Method for detecting MicroRNA 145 by adopting fluorescent probe

Abstract

The invention relates to the technical field of biological detection, in particular to a method for detecting MicroRNA 145 by adopting a fluorescent probe. The nucleotide sequence of the MicroRNA 145 is as shown in SEQ ID NO: 1, and the cDNA sequence corresponding to the MicroRNA 145 is as shown in SEQ ID NO: 2. The invention relates to a PCR (Polymerase Chain Reaction) amplification primer of MicroRNA 145. The nucleotide sequence of the primer is shown as SEQ ID NO: 3 and SEQ ID NO: 4; the invention relates to a detection probe of MicroRNA 145, the nucleotide sequence of the probe is shown as SEQ ID NO: 5, the 5'end of the probe is marked with a fluorescence reporter group ROX, and the 3 'end of the probe is marked with a fluorescence quenching group MGB; according to the invention, the expression condition of the MicroRNA 145 in an organism can be accurately and rapidly detected, and a new gene detection means can be provided for diagnosis and classification of colorectal cancer, lung cancer and breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting MicroRNA 145 by using a fluorescent probe. Background Art

[0002] Colorectal cancer, lung cancer and breast cancer are common malignant tumors. Although current detection methods have made certain progress, they still have some limitations. Colonoscopy is the gold standard for colorectal cancer screening and can directly observe lesions in the intestine. However, it is invasive and the process is not suitable for everyone, especially the elderly or patients with complications. In addition, colonoscopy may lead to false negative or false positive results due to improper operation; CTC scanning can be used as an alternative to colonoscopy, but it has high radiation risks and false positive rates. The use of tumor markers in the blood such as CEA and CA19-9 for detection can avoid the above problems, but the specificity and sensitivity of these markers are limited and cannot completely replace other detection methods. The above problems also exist in lung cancer and breast cancer examinations.

[0003] MicroRNA is a class of non-coding small RNA molecules, usually between 20 and 24 nucleotides in length. They play a key role in gene expression regulation, mainly by binding to the mRNA of the target gene, thereby inhibiting its translation or promoting its degradation, and then regulating the gene expression level. Through the study of MicroRNAs expression profiles in colorectal cancer and surrounding mucosal tissues, it was found that the expression level of MicroRNA 145, GeneBank number: NR_029686.1 in tumor tissues was significantly lower than that in normal tissues. In subsequent studies, it was found that MicroRNA 145 was closely related to the occurrence of tumors. Using MicroRNA 145 as a molecular marker to detect colorectal cancer, lung cancer and breast cancer is a detection method with accurate results, fast operation and no risks. Therefore, it is of great significance to develop a method using MicroRNA 145 as a molecular marker to detect colorectal cancer, lung cancer and breast cancer.

[0004] In view of the above problems, the present invention provides a solution. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting MicroRNA 145 using a fluorescent probe, which can accurately and quickly detect the expression of MicroRNA 145 in an organism and provide a new gene detection method for the diagnosis of colorectal cancer, lung cancer and breast cancer.

[0006] To achieve the above object, the present invention adopts the following technical scheme: a MicroRNA 145 whose nucleotide sequence is shown in SEQ ID NO: 1, and its corresponding cDNA sequence is shown in SEQ ID NO: 2;

[0007] Furthermore, the present invention provides a PCR amplification primer for MicroRNA 145, the nucleotide sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:4, the primer shown in SEQ ID NO:3 is a forward PCR primer for amplifying the cDNA corresponding to MicroRNA 145, and the primer shown in SEQ ID NO:4 is a reverse PCR primer for amplifying the cDNA corresponding to MicroRNA 145;

[0008] Furthermore, the present invention provides a detection probe for MicroRNA 145, the nucleotide sequence of the probe is shown in SEQ ID NO: 5, the 5' end of the probe is labeled with a fluorescent reporter group ROX, and the 3' end of the probe is labeled with a fluorescent quencher group MGB;

[0009] Furthermore, the present invention provides a detection kit for MicroRNA 145, wherein the nucleotide sequence of MicroRNA 145 is shown in SEQ ID NO: 1, and the corresponding cDNA sequence is shown in SEQ ID NO: 2;

[0010] The detection kit includes a PCR amplification primer for MicroRNA 145, the nucleotide sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:4, the primer shown in SEQ ID NO:2 is a forward PCR primer for amplifying the cDNA corresponding to MicroRNA 145, and the primer shown in SEQ ID NO:3 is a reverse PCR primer for amplifying the cDNA corresponding to MicroRNA 145;

[0011] The kit includes a detection probe for MicroRNA 145, the nucleotide sequence of the probe is shown in SEQ ID NO: 5, the 5' end of the probe is labeled with a fluorescent reporter group ROX, and the 3' end of the probe is labeled with a fluorescent quencher group MGB;

[0012] Furthermore, the kit includes an RNA extraction reagent, a reverse transcription reaction system, a genomic DNA reaction system and a PCR reaction solution;

[0013] Furthermore, the present invention provides a method for detecting MicroRNA 145 using a fluorescent probe, comprising the following steps:

[0014] S1: Draw 5 ml of venous blood in a fasting state into a blood collection tube without anticoagulant. After standing for 30 minutes, place the blood sample in a centrifuge and centrifuge at 1000 rpm / min for 5-10 minutes. Take the supernatant and transfer it to an enzyme-free EP tube. Centrifuge it at 1000 rpm / min for 2-4 minutes. Take the supernatant and transfer it to a new enzyme-free EP tube to obtain a serum sample, which is stored at -80°C for later use.

[0015] S2: Take 1 ml of serum sample into an EP tube, add 2 ml of Trizol, mix by pipetting repeatedly, let stand on ice for 10 min, add 200 ul of chloroform after standing, mix and shake until uniform, let stand on ice for 10 min;

[0016] S3: After standing, place the sample in a centrifuge, centrifuge at 4°C, 10,000 rpm / min for 10 min, carefully aspirate the supernatant, quickly transfer the supernatant to a new enzyme-free 1.5 ml EP tube, then add 0.5 ml of isopropanol, mix well, and stand on ice for 10 min;

[0017] S4: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 min. After centrifugation, discard the supernatant and add 1 ml of alcohol, centrifuge at 4°C, 10,000 rpm / min for 10 min. After centrifugation, discard the supernatant, dry it, and add 30-100 ul of DEPC to dissolve it.

[0018] S5: Use DNA digestion enzyme to remove the residual genomic DNA. The total volume of the reaction solution is 10 ml. The reaction solution is digested at 37°C for 40 min and then at 85°C for 3 min to inactivate the DNA digestion enzyme.

[0019] The specific reaction system is as follows:

[0020]

[0021]

[0022] S6: RNA was reverse transcribed to obtain cDNA according to the reverse transcription system, and the reaction program was 37°C for 15 min, 42°C for 25 min, 85°C for 4 min, and 4°C for 2 min;

[0023] The specific reaction system is as follows:

[0024]

[0025] S7: amplifying the obtained cDNA by fluorescence quantitative PCR;

[0026] The specific reaction system is as follows:

[0027]

[0028] The PCR amplification program is as follows, with a total of 45 cycles of amplification-denaturation, amplification-annealing and amplification-extension:

[0029]

[0030]

[0031] S8: By analyzing the fluorescence signal amplified by S7 fluorescence quantitative PCR, the expression of MicroRNA145 in the test sample can be determined to diagnose colorectal cancer, lung cancer and breast cancer and further stage and classify them.

[0032] In summary, due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: the present invention provides a MicroRNA molecule, MicroRNA 145, and uses the MicroRNA molecule as a molecular marker to diagnose colorectal cancer, lung cancer and breast cancer. At the same time, the present invention also provides a kit for diagnosing colorectal cancer, lung cancer and breast cancer using the cyclic molecule as a marker, which can accurately and quickly detect the expression of MicroRNA 145 in the organism, and can provide a new gene detection method for the diagnosis and classification of colorectal cancer, lung cancer and breast cancer. DETAILED DESCRIPTION

[0033] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0034] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0035] Example 1

[0036] 1: Disinfect the operating table with ultraviolet light for 30 minutes, then prepare the culture medium 44.5ml DMEM+5ml FBS+0.5ml double antibody, mix ampicillin, kanamycin and streptomycin at a concentration of 1 / 1000 to make triple antibody, take the foreskin of a healthy person and soak it in 75% disinfectant alcohol, wash it for 15 minutes, then take out the human foreskin tissue and transfer it to the triple antibody and soak it for 30 minutes;

[0037] 2: Transfer the soaked human foreskin tissue to sterile water and soak and wash for 15 minutes. Put the soaked human foreskin tissue into the culture medium. Use a sterile glass slide to press the human foreskin tissue to the bottom of the culture dish and culture it normally. Observe it once a day, change the culture medium every two days, and wait for the foreskin cells to crawl out from the bottom of the culture dish.

[0038] 3: On the 16th day, when a large number of foreskin cells were found at the bottom of the culture dish, the human foreskin was removed and the foreskin cells were digested and passaged using 9% Tris-EDTA trypsin. The foreskin cell suspension was pipetted into a 15 ml centrifuge tube, 6 ml of DMEM (+ / +) was added, the tube was balanced and centrifuged at 1000 rpm / min for 3 minutes. After the centrifugation, the supernatant was discarded, 1 ml of culture medium was added to resuspend the cells, and the resuspended cells were evenly spread in a 60 mm culture dish. The dish was evenly shaken up and down and left and right, and then the culture dish was placed in a cell culture incubator with 5% CO2 for culture;

[0039] 4: Turn on the water bath and heat to 37°C. Then use tweezers to quickly take out the Hela cells from the liquid nitrogen and immediately place them in a water bath to thaw. Pipette the Hela cell suspension into a 15ml centrifuge tube, add 6ml DMEM (+ / +), balance and centrifuge at 1000rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1ml culture medium to resuspend the cells, evenly spread the resuspended cells in a 60mm culture dish, shake the dish evenly up and down and left and right, and then place the culture dish in a cell culture incubator with 5% CO2 for culture;

[0040] 5: When the cell density reaches 85% under the microscope, the original culture medium is discarded, the cells are washed twice with PBS, and the foreskin cells and Hela cells are digested with 9% Tris-EDTA trypsin. The foreskin cell suspension and Hela cells are respectively pipetted into two 15ml centrifuge tubes, 6ml DMEM (+ / +) is added, balanced and centrifuged at 1000rpm / min for a total of 3 minutes. After the centrifugation is completed, the supernatant is discarded, 1ml Trizol is added to each tube, and the tube is allowed to stand at room temperature for 5min, then transferred to two new enzyme-free 1.5ml EP tubes, and allowed to stand on ice for 10min. Subsequently, 200ul chloroform is added to each tube, mixed and shaken until uniform, and allowed to stand on ice for 10min. After standing, the sample is placed in a centrifuge, centrifuged at 4℃, 10000rpm / min for 10min, and the supernatant is carefully aspirated. The supernatant is quickly transferred to two new enzyme-free 1.5ml EP tubes, and then 0.5ml isopropanol is added to each tube, mixed and allowed to stand on ice for 10min.

[0041] 5: After standing, centrifuge for 15 minutes at 4℃10000rpm / min. After centrifugation, discard the supernatant, add 1ml of alcohol, centrifuge for 10 minutes at 4℃10000rpm / min, discard the supernatant, dry, and add 50ul of DEPC to dissolve;

[0042] 6: Use DNA digestion enzymes to remove the residual genomic DNA. The total volume of the reaction solution is 10 ml. The reaction solution is digested at 37°C for 40 minutes and then at 85°C for 3 minutes to inactivate the DNA digestion enzymes.

[0043] The reaction system is the same, as follows:

[0044]

[0045] 7: Reverse transcribe the RNA to obtain cDNA according to the reverse transcription system. The reaction procedure is 37℃15min, 42℃25min, 85℃4min, 4℃2min;

[0046] The reaction system is the same, as follows:

[0047]

[0048] 8: Amplify the obtained cDNA by fluorescence quantitative PCR;

[0049] The reaction system is the same, as follows:

[0050]

[0051] The PCR amplification program is as follows, with a total of 45 cycles of amplification-denaturation, amplification-annealing and amplification-extension:

[0052]

[0053] 9: By analyzing the fluorescence signal amplified by fluorescence quantitative PCR, it can be seen that the fluorescence signal of foreskin cells is 4 times that of Hela cells. This shows that compared with normal human foreskin cells, the expression level of MicroRNA 145 in tumor cells Hela is significantly lower.

[0054] Example 2

[0055] 100 volunteers aged ≥18 years with no history of genetic diseases were recruited, including 42 normal volunteers, 18 colorectal cancer patients, 21 lung cancer patients, and 19 breast cancer patients for sample collection;

[0056] 5 ml of venous blood was drawn from the fasting state into a blood collection tube without anticoagulant. After standing for 30 minutes, the blood sample was placed in a centrifuge and centrifuged at 1000 rpm / min for 10 minutes. The supernatant was transferred to an enzyme-free EP tube and then centrifuged at 1000 rpm / min for 4 minutes. The supernatant was transferred to a new enzyme-free EP tube to obtain a serum sample, which was stored at -80°C for later use.

[0057] The volunteer serum samples were subjected to the following operations;

[0058] 1: Take 1 ml of serum sample into an EP tube, add 2 ml of Trizol, mix by pipetting repeatedly, let stand on ice for 10 min, add 200 ul of chloroform after standing, mix and shake until uniform, let stand on ice for 10 min;

[0059] 2: After standing, place the sample in a centrifuge, centrifuge at 4°C, 10,000 rpm / min for 10 min, carefully aspirate the supernatant, quickly transfer the supernatant to a new enzyme-free 1.5 ml EP tube, then add 0.5 ml of isopropanol, mix well, and stand on ice for 10 min;

[0060] 3: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 min. After centrifugation, discard the supernatant and add 1 ml of alcohol. Centrifuge at 4°C, 10,000 rpm / min for 10 min. After centrifugation, discard the supernatant, dry it, and add 100 ul of DEPC to dissolve it.

[0061] 4: Use DNA digestion enzyme to remove the residual genomic DNA. The total volume of the reaction solution is 10 ml. The reaction solution is digested at 37°C for 40 minutes and then at 85°C for 3 minutes to inactivate the DNA digestion enzyme.

[0062] The specific reaction system is as follows:

[0063]

[0064] 5: Reverse transcribe the RNA to obtain cDNA according to the reverse transcription system. The reaction procedure is 37℃15min, 42℃25min, 85℃4min, 4℃2min;

[0065] The specific reaction system is as follows:

[0066]

[0067]

[0068] 6: Amplify the obtained cDNA by fluorescence quantitative PCR;

[0069] The specific reaction system is as follows:

[0070]

[0071] The PCR amplification program is as follows, with a total of 45 cycles of amplification-denaturation, amplification-annealing and amplification-extension:

[0072]

[0073] 7: The expression level of MicroRNA 145 was obtained by analyzing the fluorescence signal of quantitative PCR amplification, and the following results were obtained using GAPDH as the control gene;

[0074] Table 1. Relative expression levels of MicroRNA 145 in volunteers

[0075] Grouping Relative expression level of MicroRNA145 Normal Volunteer 1.91±0.23 Colorectal cancer patients 0.64±0.54 Lung cancer patients 0.87±0.62 Breast cancer patients 0.94±0.63

[0076] By analyzing Table 1, it can be seen that the relative expression level of MicroRNA 145 in serum samples of normal volunteers is 1.21±0.13, and the relative expression level of MicroRNA 145 in serum samples of colorectal cancer patients, lung cancer patients and breast cancer patients is 0.3-0.5 times the relative expression level of MicroRNA 145 in serum samples of normal volunteers. Compared with normal volunteers, the expression level of MicroRNA 145 in colorectal cancer patients, lung cancer patients and breast cancer patients is significantly reduced.

[0077] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A MicroRNA 145, characterized in that: The nucleotide sequence is shown in SEQ ID NO:1, and the corresponding cDNA sequence is shown in SEQ ID NO:

2.

2. A PCR amplification primer for MicroRNA 145, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:

4. The primer shown in SEQ ID NO:3 is a forward PCR primer for amplifying the cDNA corresponding to MicroRNA 145, and the primer shown in SEQ ID NO:4 is a reverse PCR primer for amplifying the cDNA corresponding to MicroRNA 145.

3. A detection probe for MicroRNA 145, characterized in that: The nucleotide sequence of the probe is shown in SEQ ID NO:

5. The 5' end of the probe is labeled with a fluorescent reporter group ROX, and the 3' end of the probe is labeled with a fluorescent quencher group MGB.

4. A detection kit for MicroRNA 145, characterized in that: The nucleotide sequence of MicroRNA 145 is shown in SEQ ID NO: 1, and the corresponding cDNA sequence is shown in SEQ ID NO:

2.

5. A detection kit for MicroRNA 145 according to claim 4, characterized in that: The kit includes a PCR amplification primer for MicroRNA 145, the nucleotide sequence of the primer is shown in SEQ ID NO:3 and SEQ ID NO:4, the primer shown in SEQ ID NO:3 is a forward PCR primer for amplifying the cDNA corresponding to MicroRNA 145, and the primer shown in SEQ ID NO:4 is a reverse PCR primer for amplifying the cDNA corresponding to MicroRNA 145.

6. A MicroRNA 145 detection kit according to claim 4, characterized in that: The kit includes a detection probe for MicroRNA 145, the nucleotide sequence of the probe is shown in SEQ ID NO: 5, the 5' end of the probe is labeled with a fluorescent reporter group ROX, and the 3' end of the probe is labeled with a fluorescent quencher group MGB.

7. A MicroRNA 145 detection kit according to claim 4, characterized in that: The kit comprises an RNA extraction reagent, a reverse transcription reaction system, a genomic DNA reaction system and a PCR reaction solution.

8. A method for detecting MicroRNA 145 using a fluorescent probe, characterized in that: The following steps are involved: S1: Draw 5 ml of venous blood in a fasting state into a blood collection tube without anticoagulant. After standing for 30 minutes, place the blood sample in a centrifuge and centrifuge at 1000 rpm / min for 5-10 minutes. Take the supernatant and transfer it to an enzyme-free EP tube. Centrifuge it at 1000 rpm / min for 2-4 minutes. Take the supernatant and transfer it to a new enzyme-free EP tube to obtain a serum sample, which is stored at -80°C for later use. S2: Take 1 ml of serum sample into an EP tube, add 2 ml of Trizol, mix by pipetting repeatedly, let stand on ice for 10 min, add 200 ul of chloroform after standing, mix and shake until uniform, let stand on ice for 10 min; S3: After standing, place the sample in a centrifuge, centrifuge at 4°C, 10,000 rpm / min for 10 min, carefully aspirate the supernatant, quickly transfer the supernatant to a new enzyme-free 1.5 ml EP tube, then add 0.5 ml of isopropanol, mix well, and stand on ice for 10 min; S4: After standing, transfer to a centrifuge and centrifuge at 4°C, 10,000 rpm / min for 15 min. After centrifugation, discard the supernatant and add 1 ml of alcohol, centrifuge at 4°C, 10,000 rpm / min for 10 min. After centrifugation, discard the supernatant, dry it, and add 30-100 ul of DEPC to dissolve it; S5: Use DNA digestion enzyme to remove the residual genomic DNA. The total volume of the reaction solution is 10 ml. The reaction solution is digested at 37°C for 40 min and then at 85°C for 3 min to inactivate the DNA digestion enzyme. The specific reaction system is as follows: S6: Reverse transcription of RNA was performed to obtain cDNA according to the reverse transcription system. The reaction procedure was 37°C for 15 min, 42°C for 25 min, 85°C for 4 min, and 4°C for 2 min. The specific reaction system is as follows: S7: amplifying the obtained cDNA by fluorescence quantitative PCR; The specific reaction system is as follows: The PCR amplification program is as follows, with a total of 45 cycles of amplification-denaturation, amplification-annealing and amplification-extension: S8: The expression of MicroRNA 145 in the test sample can be determined by analyzing the fluorescent signal amplified by the fluorescent quantitative PCR of S7.

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