CN117418007A - Application of COX7CmRNA detection reagent in preparation of MM screening kit and kit - Google Patents
Application of COX7CmRNA detection reagent in preparation of MM screening kit and kit Download PDFInfo
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 65
- 238000001514 detection method Methods 0.000 title claims abstract description 42
- 238000012216 screening Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 53
- 208000034578 Multiple myelomas Diseases 0.000 claims abstract description 37
- 210000004180 plasmocyte Anatomy 0.000 claims abstract description 16
- 238000011529 RT qPCR Methods 0.000 claims description 13
- 238000000636 Northern blotting Methods 0.000 claims description 8
- 210000000988 bone and bone Anatomy 0.000 claims description 8
- 108020004999 messenger RNA Proteins 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 238000012165 high-throughput sequencing Methods 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 abstract description 14
- 201000010099 disease Diseases 0.000 abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 12
- 108060003951 Immunoglobulin Proteins 0.000 abstract description 11
- 102000018358 immunoglobulin Human genes 0.000 abstract description 11
- 201000000050 myeloid neoplasm Diseases 0.000 description 16
- 102100030512 Cytochrome c oxidase subunit 7C, mitochondrial Human genes 0.000 description 7
- 101000919491 Homo sapiens Cytochrome c oxidase subunit 7C, mitochondrial Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000003745 diagnosis Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 239000011324 bead Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108020005196 Mitochondrial DNA Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000002407 ATP formation Effects 0.000 description 1
- 206010006002 Bone pain Diseases 0.000 description 1
- 101710085938 Matrix protein Proteins 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 208000010190 Monoclonal Gammopathy of Undetermined Significance Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000007452 Plasmacytoma Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 230000024245 cell differentiation Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 201000005328 monoclonal gammopathy of uncertain significance Diseases 0.000 description 1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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Abstract
The invention belongs to the field of bioscience, and in particular relates to a biological preparation methodCOX7C mRNAApplication of detection reagent in preparing MM screening kit and kit. The invention discovers the bone marrow plasma cells of the patients with the multiple myeloma for the first timeCOX7C mRNAIs significantly higher than in the case of the unknown monoclonal immunoglobulin disease (MGUS) patients and healthy controls. The invention will detectCOX7C mRNAThe reagent of the level is used for preparing a multiple myeloma screening kit, so that the multiple myeloma can be effectively screened.
Description
Technical Field
The invention belongs to the field of medical science, in particular to a medicine compositionCOX7CmRNAApplication of detection reagent in preparing MM screening kit and kit.
Background
Multiple Myeloma (MM) is a malignant plasma cytopathy characterized by abnormal proliferation of plasma cells in the bone marrow. The proliferated plasma cells produce monoclonal immunoglobulin, which causes damage to target organs, and causes bad conditions such as anemia, bone pain, bone destruction, and kidney function impairment, thus bringing heavy burden to the family and society of patients. The incidence rate of MM in China is about one ten thousandth, and the MM accounts for about 1% of all cancers.
Multiple myeloma is not clearly distinguished clinically from the unknown monoclonal immunoglobulin disease (MGUS), especially in the early stages of the disease. Meanwhile, due to the high heterogeneity of myeloma, diagnosis also becomes quite complex, bringing a great economic burden to patients and their families. Therefore, the development of a novel and effective myeloma diagnosis method has extremely important significance for early diagnosis of diseases, improvement of treatment effects and improvement of life quality of patients.
COX7CmRNAIs an mRNA in mitochondria that encodes a protein that is part of the complex in mitochondria involved in ATP synthesis, i.e., COX7C.COX7CmRNACan be used as termination signal of mitochondrial DNA transcription to control the program and degree of mitochondrial gene expression. In addition, in the case of the optical fiber,COX7CmRNAcan also be involved in apoptosis and cell differentiation.
At present not yet seeCOX7CmRNAPrior art related to multiple myeloma.
Disclosure of Invention
The invention aims to provideCOX7CmRNAPreparation of detection reagentApplication of the kit in preparing MM screening kit and the kit.
The invention provides a detection methodCOX7CmRNAUse of a level of a reagent in the preparation of a multiple myeloma screening kit.
Further, the detectingCOX7CmRNAThe level reagent comprises a real-time quantitative PCR detection reagent, a Northern Blot detection reagent, a high-throughput sequencing reagent, a gene chip reagent or a nucleic acid Blot detection reagent.
Further, the detectingCOX7CmRNAThe horizontal reagent is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent comprises a primer sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
Further, the detectingCOX7CmRNAThe reagent at the level is a Northern Blot detection reagent.
Further, the detectingCOX7CmRNAThe level of the reagent is detected in plasma cells in human bone marrowCOX7CmRNAIs a reagent of (a).
The invention also provides a multiple myeloma screening kit which comprises the kit for detectingCOX7CmRNAIs a reagent of (a).
Further, the detectingCOX7CmRNAThe level reagent comprises a real-time quantitative PCR detection reagent, a Northern Blot detection reagent, a high-throughput sequencing reagent, a gene chip reagent or a nucleic acid Blot detection reagent.
Further, the detectingCOX7CmRNAThe horizontal reagent is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent comprises a primer sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
Further, the detectingCOX7CmRNAThe reagent at the level is a Northern Blot detection reagent.
Further, the detectingCOX7CmRNAThe level of the reagent is detected in plasma cells in human bone marrowCOX7C mRNAA level of reagent.
The invention has the beneficial effects that: the kit of the invention can detect the bone marrowCOX7CmRNACan screen the risk degree of multiple myeloma of the group to be examined: if it isCOX7CmRNALow levels, with multiple sexesLow risk of myeloma, ifCOX7CmRNAHigh levels, the risk of multiple myeloma is high. The invention will detectCOX7CmRNAThe reagent of the kit is used for preparing a multiple myeloma screening kit, has high specificity and high sensitivity, and can realize the effective screening of multiple myeloma. The invention provides effective basis for patients to take relevant therapeutic measures or decisions, and has good clinical application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 shows a patient with multiple myeloma and an unknown monoclonal immunoglobulin disease (MGUS)COX7C mRNAHorizontal results plot.
FIG. 2 is a graph showing the relationship between healthy controls (NC), unidentified monoclonal immunoglobulin diseases (MGUS) and Multiple Myeloma (MM) in GSE6477 databaseCOX7CmRNAIs a horizontal drawing of (a).
Fig. 3 is a ROC graph of healthy controls (NC) in GSE6477 database as control.
Figure 4 ROC profile of unknown monoclonal immunoglobulin disease (MGUS) in gse6477 database as control.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
MGUS refers to an unknown monoclonal immunoglobulin disease (Monoclonal Gammopathy of Undetermined Significance). This is a benign plasmacytoma characterized by low levels of M protein in the blood, but without causing significant signs or symptoms. MGUS may develop multiple myeloma.
Example 1 real-time quantitative PCR (qPCR) detection of bone marrow plasma cellsCOX7CmRNARelationship of level to multiple myeloma
1. Experimental method
Sample collection and processing
1. Sample collection
The study subjects were bone marrow samples collected from 7 patients with unknown monoclonal immunoglobulin disease (MGUS) and 12 patients with multiple myeloma. Diagnosis of MGUS and multiple myeloma was diagnosed and confirmed by diagnostic criteria according to International Myeloma Working Group (IMWG) guidelines (version 2014) and chinese multiple myeloma diagnosis guidelines (revised 2022), both recruited from the people hospitals in the province of the Sichuan province. Basic information of the subjects is shown in table 1:
table 1 basic information of subjects
| Basic information | MGUS | Multiple myeloma |
| Number of people (n) | 7 | 12 |
| Average age (Y) | 43 | 67 |
| Male proportion (%) | 57.1 | 75 |
2. Experimental treatment
2.1 bone marrow sample collection
1-2ml of patient bone marrow is collected by bone marrow puncture under the conventional sterile condition and is placed in an EDTA anticoagulated purple head blood collection tube, and the mixture is inverted and uniformly mixed.
2.2 bone marrow plasma cell separation
To the EDTA anticoagulated bone marrow samples, 20. Mu.L of CD138+ magnetic bead reagent (Meitianfu Biotech Co.) was added to label the plasma cells, and the mixture was placed in a refrigerator at 4℃for 15min.
The magnetic bead sorting column is arranged on a sorter, 3ml of PBS buffer solution is added to the magnetic bead sorting column, and after PBS completely passes through the sorting column, incubated samples are added for sorting. The addition of 3ml of PBS buffer was repeated twice.
Taking down the separation column, adding 3ml PBS buffer solution, rapidly pushing the separation column plunger core rod for eluting, repeating the eluting for 3 times, and loading the eluent into a 10ml centrifuge tube for centrifugation (3000 r/min,10 min). After centrifugation 200. Mu.l of supernatant was kept with pellet, mixed and loaded into a 1.5ml EP tube, and the resulting sample was plasma cells isolated from bone marrow.
(II) mRNA nucleic acid extraction and amplification
mRNA of bone marrow plasma cells isolated in the above step was extracted using TRIzol, and mRNA concentration and purity were determined.
mRNA was synthesized into cDNA using a reverse transcription kit for mRNA whose concentration and purity were satisfactory.
Quantitative amplification analysis was performed on the synthesized cDNA using qPCR kit.
Use 2 -ΔΔCq The method calculates the relative expression level.
The primer sequences for COX7C were as follows:
COX7C Forward 5'-3' (SEQ ID NO. 1): CCGTAGGAGCCACTATGAGG
COX7C reverse 5'-3' (SEQ ID No. 2): GGCTGCACCTCTTAAAATGC
2. Experimental results
Multiple myeloma patients and unknown monoclonal immunoglobulin diseases (MGUS)COX7CmRNAThe results of the horizontal experiments are shown in FIG. 1. It can be seen that isolated plasma cell (CD138+) detection in patients with multiple myelomaCOX7C mRNAIs significantly higher than the isolated plasma cell (CD138+) detection of patients with monoclonal immunoglobulin diseases (MGUS) of unknown significanceCOX7C mRNAIs a level of (c).
Experimental results show that the invention detects plasma cells separated from patients through experimentsCOX7CmRNAAt a level of (1) confirmingCOX7CmRNACan be used as a marker of multiple myeloma and can provide effective basis for patients to take relevant therapeutic measures or decisions.
EXAMPLE 2,COX7CmRNAExpression in GSE6477 (database)
1. Experimental method
Analysis of myeloma patients by selection of myeloma study data in a databaseCOX7CmRNAHorizontal condition. Data are derived from the data GSE6477 of the GEO (Gene Expression Omnibus) database of NCBI. Study analysis between healthy control (NC), unidentified monoclonal immunoglobulin disease (MGUS) and Multiple Myeloma (MM)COX7C mRNAIs a difference in expression of (a).
2. Experimental results
Analysis using GSE6477 databaseCOX7CmRNAThe horizontal results are shown in fig. 2. Healthy control group (NC) 12 persons, MGUS group total 22 persons, multiple myeloma group 49 persons in GSE6477 database.
In bone marrow plasma cells of patients with multiple myelomaCOX7CmRNAAverage level of 13.56 in healthy control (NC) bone marrowCOX7CmRNAThe average level of (2) was 11.39. Multiple myeloma group and healthy control group (p<0.0001 Is statistically significant. The specificity of ROC analysis results of the multiple myeloma group and the healthy control group is 100 percent, and the sensitivity is 95.92 percent, which shows thatCOX7CmRNAMultiple myeloma can be distinguished specifically from healthy controls, as shown in figure 3.
In bone marrow of patients with MGUSCOX7CmRNAThe average level of (2) was 11.64. Multiple myeloma group and MGUS group (p)<0.0001 Is statistically significant. The specificity of ROC analysis results of the multiple myeloma group and the healthy control group is 100 percent, the sensitivity is 95.92 percent,indicating thatCOX7CmRNAMultiple myeloma can be distinguished specifically from MGUS as shown in figure 4. The experimental result shows that the method has the advantages of high yield,COX7CmRNAcan be used for the auxiliary diagnosis of clinical multiple myeloma.
The invention is combined with experiments and database analysis to prove that the bone marrow plasma cells of the patients with the multiple myelomaCOX7CmRNAThe level is significantly higher than normal and MGUS patients,COX7C mRNAcan be used as a marker of multiple myeloma, and provides effective basis for patients to take relevant therapeutic measures or decisions.
In summary, the kit of the invention can detect the bone marrowCOX7C mRNACan screen the risk degree of multiple myeloma of the group to be examined: if it isCOX7CmRNALow levels, the risk of multiple myeloma is low ifCOX7CmRNAHigh levels, the risk of multiple myeloma is high. The invention will detectCOX7CmRNAThe reagent of the kit is used for preparing a multiple myeloma screening kit, has high specificity and high sensitivity, and can realize the effective screening of multiple myeloma. The invention provides effective basis for patients to take relevant therapeutic measures or decisions, and has good clinical application prospect.
Claims (10)
1. Detection ofCOX7CmRNAUse of a level of a reagent in the preparation of a multiple myeloma screening kit.
2. The use according to claim 1, wherein the detection isCOX7CmRNAThe level reagent comprises a real-time quantitative PCR detection reagent, a Northern Blot detection reagent, a high-throughput sequencing reagent, a gene chip reagent or a nucleic acid Blot detection reagent.
3. The use according to claim 1, wherein the detection isCOX7CThe reagent of mRNA level is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent comprises a primer sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
4. Use according to claim 1Characterized in that the detectionCOX7CmRNAThe reagent at the level is a Northern Blot detection reagent.
5. The use according to any one of claims 1 to 4, wherein the detection is performed byCOX7CmRNAThe level of the reagent is detected in plasma cells in human bone marrowCOX7CmRNAIs a reagent of (a).
6. A multiple myeloma screening kit comprising a reagent for detectingCOX7CmRNAIs a reagent of (a).
7. The kit of claim 6, wherein the detection is performed byCOX7CmRNAThe level reagent comprises a real-time quantitative PCR detection reagent, a Northern Blot detection reagent, a high-throughput sequencing reagent, a gene chip reagent or a nucleic acid Blot detection reagent.
8. The kit of claim 6, wherein the detection is performed byCOX7CmRNAThe horizontal reagent is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent comprises a primer sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
9. The kit of claim 6, wherein the detection is performed byCOX7CmRNAThe reagent at the level is a Northern Blot detection reagent.
10. The kit of any one of claims 6 to 8, wherein the detection is performed byCOX7CmRNAThe level of the reagent is detected in plasma cells in human bone marrowCOX7CmRNAA level of reagent.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080280779A1 (en) * | 2006-09-26 | 2008-11-13 | Shaughnessy Jr John D | Gene expression profiling based identification of genomic signatures of multiple myeloma and uses thereof |
| US20140357512A1 (en) * | 2013-06-03 | 2014-12-04 | Acetylon Pharmaceuticals, Inc. | Histone deacetylase (hdac) biomarkers in multiple myeloma |
| CN110232974A (en) * | 2019-04-22 | 2019-09-13 | 福建医科大学附属第一医院 | A kind of novel Huppert's disease integrated risk methods of marking |
| US20230272484A1 (en) * | 2020-11-03 | 2023-08-31 | Yeda Research And Development Co. Ltd. | Methods of prognosing, determining treatment course and treating multiple myeloma |
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- 2023-10-20 CN CN202311366927.2A patent/CN117418007A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080280779A1 (en) * | 2006-09-26 | 2008-11-13 | Shaughnessy Jr John D | Gene expression profiling based identification of genomic signatures of multiple myeloma and uses thereof |
| US20140357512A1 (en) * | 2013-06-03 | 2014-12-04 | Acetylon Pharmaceuticals, Inc. | Histone deacetylase (hdac) biomarkers in multiple myeloma |
| CN110232974A (en) * | 2019-04-22 | 2019-09-13 | 福建医科大学附属第一医院 | A kind of novel Huppert's disease integrated risk methods of marking |
| US20230272484A1 (en) * | 2020-11-03 | 2023-08-31 | Yeda Research And Development Co. Ltd. | Methods of prognosing, determining treatment course and treating multiple myeloma |
Non-Patent Citations (2)
| Title |
|---|
| MAGNE BØRSET等: "Highly expressed genes in multiple myeloma cells –what can they tell us about the disease?", EUR J HAEMATOL, vol. 109, 31 December 2022 (2022-12-31), pages 31 - 40 * |
| SAMRAT ROY CHOUDHURY等: "The functional epigenetic landscape of aberrant gene expression in molecular subgroups of newly diagnosed multiple myeloma", JOURNAL OF HEMATOLOGY & ONCOLOGY, vol. 13, 31 December 2020 (2020-12-31), pages 108, XP055796906, DOI: 10.1186/s13045-020-00933-y * |
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