New diagnostic function of MAEA gene in blood

Abstract

The invention discloses an MAEA gene and an application of an expression product of the MAEA gene to preparation of a diagnostic product. The diagnostic product can be used for diagnosing myocardial infarction. The risk of myocardial infarction of a testee in the future can be judged or the state of myocardial infarction is confirmed by detecting the level of the MAEA gene and the expression product thereof. The diagnostic product takes peripheral blood of the testee as a detection object, and can achieve noninvasive, rapid, sensitive and accurate diagnostic effects.

Description

technical field

[0001] The invention belongs to the field of molecular diagnosis and relates to a new diagnostic function of MAEA gene or protein. Background technique

[0002] Myocardial infarction is a global public health problem that seriously threatens public health (J.Ross, Jr., A50-year research journey. From laboratory to clinic, Circulation journal: official journal of the Japanese Circulation Society, 73(2009 ) 3-12; D. Lloyd-Jones, R. Adams, M. Carnethon, G. De Simone, T. B. Ferguson, K. Flegal, E. Ford, K. Furie, A. Go, K. Greenlund, N. Haase , S. Hailpern, M. Ho, V. Howard, B. Kissela, S. Kittner, D. Lackland, L. Lisabeth, A. Marelli, M. McDermott, J. Meigs, D. Mozaffarian, G. Nichol, C . O'Donnell, V. Roger, W. Rosamond, R. Sacco, P Sorlie, R. Stafford, J. Steinberger, T. Thom, S. Wasserthiel-Smoller, N. Wong, J. Wylie-Rosett, Y. Hong, C. American Heart Association Statistics, S.). Several methodologies can be effectively applied to the treatment of myocardi...

Examples

Embodiment 1

[0034] Example 1 Screening of genes differentially expressed in patients with myocardial infarction and normal people

[0035] 1. Research object

[0036] Select 6 hospitalized patients with myocardial infarction treated in the hospital as research objects, including 3 males and 3 females, with an average age of 56.00±8.75; 7 healthy people in the control group; each of the above-mentioned patients and healthy people who were invited to participate in the study All signed the informed consent.

[0037] 2. Sample collection and storage

[0038] On the day of admission, 8 mL of fresh sterile arterial blood was collected into EDTA anticoagulated purple tubes before coronary angiography. If it is not used immediately, the sample can be stored in a refrigerator at 4°C for 2 hours.

[0039] 3. Separation of PBMCs by Ficoll method

[0040] The following steps are all completed in the ultra-clean bench:

[0041] (1) Dilute the blood sample with equal volume of normal saline, add ...

Embodiment 2

[0063] Embodiment 2QPCR experiment verifies the genes differentially expressed in patients with myocardial infarction and normal people

[0064] 1. Research object:

[0065] The screening criteria were the same as in Example 1, 35 patients with myocardial infarction and 35 normal subjects.

[0066] 2. Extraction of total RNA in blood

[0067] Step is with embodiment 1.

[0068] 3. RT-PCR

[0069] (1)RT

[0070] RT reaction system (20μl):

[0071]

[0072] RT reaction procedure:

[0073] 42°C 15min

[0074] 85℃ 5s

[0075] 4℃ ---

[0076] (2) qPCR

[0077] PCR reaction system (20μl):

[0078]

[0079] PCR reaction program:

[0080] Amplification procedure:

[0081] Stage 1 95°C 10min

[0082] Stage 2 95°C 10s

[0083] 51°C 10s

[0084] Repeat for 45 cycles

[0085] Three replicate wells were set for each sample, and the internal reference was GAPDH.

[0086] (3) Primers

[0087] The primer sequences of MAEA gene and GAPDH gene are as follows:

[0088] GAP...

Embodiment 3

[0101] Example 3 Western blot experiment to verify the expression products of differentially expressed genes in patients with myocardial infarction and normal people

[0102] 1. Research object: same as embodiment 2.

[0103] 2. Mononuclear cell isolation

[0104] Take 10ml of venous blood from patients with myocardial infarction and normal people, inject it into a sterile vial containing heparin, and shake it gently immediately after capping. Add an equal volume of HBSS (NaCl 8.0g, NaCl 2 HPO 4 0.132g, KH 2 PO 4 0.06g, KCl0.4g, phenol red 1ml, NaHCO 3 0.35g, D-glucose 1.0g, dissolved in 1000ml double distilled water), to reduce the aggregation of red blood cells. Draw 8ml of lymphocyte stratification solution into a 50ml centrifuge tube, slowly add the diluted blood along the tube wall, keep the interface clear, do not mix the two, centrifuge at 20°C 2000r / min for 30min, carefully absorb the stratification solution and plasma transfer The turbid off-white layer, tha...

technical field

[0001] The invention belongs to the field of molecular diagnosis and relates to a new diagnostic function of MAEA gene or protein. Background technique

[0002] Myocardial infarction is a global public health problem that seriously threatens public health (J.Ross, Jr., A50-year research journey. From laboratory to clinic, Circulation journal: official journal of the Japanese Circulation Society, 73(2009 ) 3-12; D. Lloyd-Jones, R. Adams, M. Carnethon, G. De Simone, T. B. Ferguson, K. Flegal, E. Ford, K. Furie, A. Go, K. Greenlund, N. Haase , S. Hailpern, M. Ho, V. Howard, B. Kissela, S. Kittner, D. Lackland, L. Lisabeth, A. Marelli, M. McDermott, J. Meigs, D. Mozaffarian, G. Nichol, C . O'Donnell, V. Roger, W. Rosamond, R. Sacco, P Sorlie, R. Stafford, J. Steinberger, T. Thom, S. Wasserthiel-Smoller, N. Wong, J. Wylie-Rosett, Y. Hong, C. American Heart Association Statistics, S.). Several methodologies can be effectively applied to the treatment of myocardi...

Examples

Embodiment 1

[0034] Example 1 Screening of genes differentially expressed in patients with myocardial infarction and normal people

[0035] 1. Research object

[0036] Select 6 hospitalized patients with myocardial infarction treated in the hospital as research objects, including 3 males and 3 females, with an average age of 56.00±8.75; 7 healthy people in the control group; each of the above-mentioned patients and healthy people who were invited to participate in the study All signed the informed consent.

[0037] 2. Sample collection and storage

[0038] On the day of admission, 8 mL of fresh sterile arterial blood was collected into EDTA anticoagulated purple tubes before coronary angiography. If it is not used immediately, the sample can be stored in a refrigerator at 4°C for 2 hours.

[0039] 3. Separation of PBMCs by Ficoll method

[0040] The following steps are all completed in the ultra-clean bench:

[0041] (1) Dilute the blood sample with equal volume of normal saline, add ...

Embodiment 2

[0063] Embodiment 2QPCR experiment verifies the genes differentially expressed in patients with myocardial infarction and normal people

[0064] 1. Research object:

[0065] The screening criteria were the same as in Example 1, 35 patients with myocardial infarction and 35 normal subjects.

[0066] 2. Extraction of total RNA in blood

[0067] Step is with embodiment 1.

[0068] 3. RT-PCR

[0069] (1)RT

[0070] RT reaction system (20μl):

[0071]

[0072] RT reaction procedure:

[0073] 42°C 15min

[0074] 85℃ 5s

[0075] 4℃ ---

[0076] (2) qPCR

[0077] PCR reaction system (20μl):

[0078]

[0079] PCR reaction program:

[0080] Amplification procedure:

[0081] Stage 1 95°C 10min

[0082] Stage 2 95°C 10s

[0083] 51°C 10s

[0084] Repeat for 45 cycles

[0085] Three replicate wells were set for each sample, and the internal reference was GAPDH.

[0086] (3) Primers

[0087] The primer sequences of MAEA gene and GAPDH gene are as follows:

[0088] GAP...

Embodiment 3

[0101] Example 3 Western blot experiment to verify the expression products of differentially expressed genes in patients with myocardial infarction and normal people

[0102] 1. Research object: same as embodiment 2.

[0103] 2. Mononuclear cell isolation

[0104] Take 10ml of venous blood from patients with myocardial infarction and normal people, inject it into a sterile vial containing heparin, and shake it gently immediately after capping. Add an equal volume of HBSS (NaCl 8.0g, NaCl 2 HPO 4 0.132g, KH 2 PO 4 0.06g, KCl0.4g, phenol red 1ml, NaHCO 3 0.35g, D-glucose 1.0g, dissolved in 1000ml double distilled water), to reduce the aggregation of red blood cells. Draw 8ml of lymphocyte stratification solution into a 50ml centrifuge tube, slowly add the diluted blood along the tube wall, keep the interface clear, do not mix the two, centrifuge at 20°C 2000r / min for 30min, carefully absorb the stratification solution and plasma transfer The turbid off-white layer, tha...

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