Use of a reagent for detecting RBP1 for the manufacture of a kit for diagnosing atrial fibrillation

Abstract

The application provides a reagent for detecting RBP1 for preparing a kit for diagnosing atrial fibrillation, difference genes of atrial fibrillation patients and non-atrial fibrillation patients are obtained through weight gene co-expression network analysis (WGCNA), and RT-qPCR is used for verification in human peripheral blood samples. Peripheral blood leukocyte extraction: 3-5ml of venous blood is taken, EDTA anticoagulation is carried out, and centrifugation is carried out at 2500rpm for 10min; the upper plasma is carefully sucked and is divided into 3 0.5ml centrifuge tubes; 3 times the volume of hemolytic liquid is added into the blood cells, and shaking is carried out, ice bath is carried out for 15min; 2500rpm centrifugation is carried out for 10min, and the supernatant is discarded. The RBP1 detection method can be used as a biomarker to assist the diagnosis of atrial fibrillation. Retinol binding protein 1 is a protein encoded by the CRABP1 gene. It binds with high affinity to retinol and retinoids, protects retinoids from nonspecific oxidation, and delivers retinoids to specific enzymes. Changes in RBP1 gene expression will affect retinoic acid production, cell proliferation and microenvironment of epithelial cells. Blood biomarker sampling is simple and low in price.

Description

Technical Field

[0001] This invention relates to the field of atrial fibrillation diagnosis, and particularly to the use of reagents for detecting RBP1 in the preparation of reagent kits for diagnosing atrial fibrillation. Background Technology

[0002] Atrial fibrillation (AF) is one of the most common cardiac arrhythmias. It is an atrial rhythm characterized by disordered excitation and ineffective contraction of the atria. It is caused by a predominant atrial reentry circuit leading to numerous smaller reentry circuits, resulting in atrial rhythm disturbances. It is very common in the elderly. AF can cause serious complications such as heart failure and stroke, leading to patient disability or increased mortality.

[0003] my country has the highest prevalence of atrial fibrillation (AF), with 7.9 million people suffering from the condition according to the latest national survey. AF can lead to complications such as myocardial infarction, heart failure, and stroke, resulting in high rates of disability and death, placing a heavy burden on society and families. However, awareness and intervention rates among AF patients in my country are very low. The latest survey data shows that as many as 36% of AF patients are unaware of their condition, and only 6% of patients with high CHA2DS2-VASc scores (≥2 for men or ≥3 for women) receive anticoagulation therapy.

[0004] According to the latest data from the National Bureau of Statistics, my country has approximately 250 million people aged 60 and above, highlighting the urgent need to improve the early diagnosis, awareness, and intervention rates for atrial fibrillation (AF). Studies have shown that widespread screening for asymptomatic AF in the elderly population can effectively improve diagnosis, awareness, and intervention rates, reducing the incidence of stroke, heart failure, and related disabilities. Currently, AF diagnosis primarily relies on electrocardiograms (ECGs) and Holter monitoring, which can easily miss cases, especially paroxysmal AF, in patients who are not experiencing an attack. In recent years, devices such as remote ECG and smart bracelets have been developed, enabling real-time transmission of ECG data for AF screening, effectively improving awareness and detection rates of asymptomatic AF. However, these devices are generally expensive. Blood biomarkers are widely used for early disease diagnosis, efficacy evaluation, and prognostic assessment due to their simple sampling and low cost; however, specific biomarkers are still lacking for AF diagnosis.

[0005] Retinol-binding protein 1 (RBP1), also known as CRABP1, is a protein encoded by the CRABP1 gene. It binds with a high affinity to retinol and retinin, protecting retinin from non-specific oxidation and delivering it to specific enzymes. Studies have found that alterations in RBP1 gene expression affect retinoic acid production, cell proliferation, and the microenvironment in epithelial cells. Summary of the Invention

[0006] The purpose of this invention is to design reagents for detecting RBP1 for use in preparing kits for diagnosing atrial fibrillation, which can serve as biomarkers for the diagnosis of atrial fibrillation.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: the use of reagents for detecting RBP1 in the preparation of reagent kits for diagnosing atrial fibrillation, characterized by comprising the following steps:

[0008] (1) Peripheral blood leukocyte extraction: Take 3-5 ml of venous blood, anticoagulate with EDTA, and centrifuge at 2500 rpm for 10 min;

[0009] (2) Carefully aspirate the upper plasma layer and dispense it into three 0.5ml centrifuge tubes;

[0010] (3) Add 3 times the volume of lysed blood to the blood cells, shake well, and incubate on ice for 15 minutes;

[0011] (4) Centrifuge at 2500 rpm for 10 min and discard the supernatant;

[0012] (5) Add 10ml of blood lysate, shake well, and incubate on ice for 15 minutes;

[0013] (6) Centrifuge at 3000 rpm for 10 min and discard the supernatant;

[0014] (7) Invert the centrifuge tube to remove any residual liquid;

[0015] (8) Obtain white blood cells and store them at -80°C to avoid repeated freezing and thawing;

[0016] (9) RNA extraction: After thawing the frozen leukocytes, add 1 ml of sterile PBS to resuspend them and centrifuge at 1000 rpm for 1 min at 4℃.

[0017] (10) After discarding the supernatant, add 1 ml of TRIzol to lyse the cells and mix by pipetting.

[0018] (11) Let stand at room temperature for 5 minutes, then transfer the supernatant to a 1.5 ml EP tube;

[0019] (12) Centrifuge at 12000 rpm for 5 min, take the supernatant, add chloroform, shake to mix, and let stand at room temperature for 15 min to allow it to separate into phases naturally;

[0020] (13) Centrifuge at 12000rpm for 15min at 4℃. The sample is divided into three layers: a yellow organic layer, a middle layer and an upper layer of colorless aqueous phase. RNA is mainly in the aqueous phase.

[0021] (13) Carefully aspirate the upper aqueous phase into a new 1.5 ml EP tube, add an equal volume of ice-cold isopropanol, and place at -20°C for 1 h;

[0022] (14) Centrifuge at 12000 rpm for 10 min at 4℃;

[0023] (15) Discard the supernatant, add 1 ml of 75% ethanol, gently shake the EP tube to suspend the precipitate;

[0024] (16) Centrifuge at 8000 rpm for 5 min at 4℃, discard the supernatant, and air dry at room temperature for 5-10 min;

[0025] (17) Dissolve the RNA precipitate in 50 μl DEPC water, detect the RNA concentration with a spectrophotometer, and store at -80℃ for later use;

[0026] (18) cDNA synthesis reaction: 4.0 μl 5× PrimeScript Buffer, 1.0 μg Total RNA, and 20 μl RNase-Free ddH2O were gently mixed and reacted at 37°C for 15 min, then reacted at 85°C for 5 s, and then gradually cooled to 4°C.

[0027] (19) RT-qPCR reaction: 10.0 μl TBGreenPremixExTaqII, 1.0 μl ForwardPrimer, 1.0 μl ReversePrimer, 2.0 μl cDNA, and 20 μl RNase-FreeddH2O were thoroughly mixed in the reaction solution. The reaction was pre-denatured at 95℃ for 30 s; denatured at 95℃ for 5 s; annealed at 60℃ for 30–34 s; for 40 cycles.

[0028] (20) The Ct value of the target gene was normalized by standardizing the Ct value of the internal reference gene GAPDH, and then 2... -△△Ct The expression level of RBP1 was obtained by performing relative quantitative analysis of gene expression differences in the sample.

[0029] As an improvement, the interval between blood collection and leukocyte separation shall not exceed 2 hours at room temperature and 5 hours at 4°C.

[0030] As an improvement, the 75% ethanol is prepared from DEPC water.

[0031] The beneficial effects of this invention are: this detection method using RBP1 as a biomarker for diagnosing atrial fibrillation can assist in the diagnosis of atrial fibrillation, especially asymptomatic and paroxysmal atrial fibrillation. Retinol-binding protein 1 (RBP1), also known as CRABP1, is a protein encoded by the CRABP1 gene. It binds with high affinity to retinol and retinin, protecting retinin from non-specific oxidation and delivering retinin to specific enzymes. Alterations in RBP1 gene expression affect retinoic acid production, cell proliferation, and the microenvironment in epithelial cells. Blood biomarker sampling is simple and inexpensive. Attached Figure Description

[0032] Figure 1 This diagram shows the application of the reagent for detecting RBP1 in the preparation of a diagnostic kit for atrial fibrillation, along with a comparison of RBP1 expression levels.

[0033] Figure 2 The ROC curve analysis diagram illustrates the diagnostic value of RBP1 for atrial fibrillation using the reagent for detecting RBP1 in this invention. Detailed Implementation

[0034] The present invention is illustrated below with specific embodiments, which are not intended to limit the scope of the invention.

[0035] The use of reagents for detecting RBP1 in the preparation of a diagnostic kit for atrial fibrillation includes the following steps:

[0036] I. Weighted Gene Co-expression Network Analysis

[0037] 1. Data Acquisition and Preprocessing

[0038] mRNA transcriptome sequencing (RNA-seq) data (database number GSE2240) was downloaded from the NCBI GEO database (Gene Expression Omnibus, https: / / www.ncbi.nlm.nih.gov / geo / ), and sequencing was performed using an Affymetrix U133 array. This dataset contains right atrial appendage data from 30 patients with permanent atrial fibrillation (AF) and sinus rhythm (SR), defined as atrial fibrillation lasting more than 3 months on ECG monitoring, and sinus rhythm maintained during cardiac surgery without prior atrial fibrillation episodes. The raw data underwent the same preprocessing, with background correction and normalization performed using R. Probes were matched to genes, and probes matching multiple genes were removed. For genes matching multiple probes, the median expression value was used as the final expression value. The SD value of each gene was calculated and sorted from highest to lowest. The top 5000 genes were selected for the construction and association analysis of the Weighted Gene Co-expression Network (WGCNA).

[0039] 2. Construction of weighted gene co-expression networks

[0040] In a gene co-expression network, nodes represent genes, and edges represent the degree of co-expression. WGCNA is based on the co-expression similarity s between the i-th and j-th genes. ij Define an adjacency matrix and calculate gene co-expression based on the adjacency matrix. Assume variable x... i s represents the expression profile of the i-th gene.ij It is usually defined as the absolute value of the Pearson correlation coefficient between the expression profiles of genes i and j, that is,

[0041] s ij =|cor(x) i x j )|

[0042] Next, co-expression similarity s ij Converted to adjacency degree 'a' via adjacency function. ij :

[0043]

[0044] Where β≥1 is a soft threshold, determined according to the scale-free topology standard.

[0045] A gene co-expression network was constructed using the WGCNA package in R. The hclust function was used to perform cluster analysis on the samples to identify outliers. The correlation between two genes was calculated using weighted correlation coefficients, and the adjacency matrix of the expression profile genes was calculated. A soft threshold β was then reasonably selected within a certain range based on the scale-free network fit index and average connectivity to ensure both scale-free network performance and good network connectivity. Setting the fit index R² > 0.85 ensured that the connections between genes followed an approximately scale-free network distribution. The pickSoftThreshold function was used to automatically select a suitable soft threshold β. The blockwiseModules function was used for network construction and module detection, generating co-expression gene modules with a minimum module size of 50 and a merge cut height of 0.25, along with a topological overlap matrix (TOM). Based on the dissimilarity of the TOM, a hierarchical clustering method using average connectivity was employed to group genes with similar expression patterns into the same module. A dynamic pruning tree method was used to identify gene modules.

[0046] 3. Correlation analysis between co-expression modules and clinical phenotypes

[0047] Gene modules are closely related gene clusters during co-expression. WGCNA uses hierarchical clustering to identify gene modules and represents them with different colors. Genes that are not assigned to any module are placed in gray modules. Principal component analysis is performed on each module, and the eigenvalues ​​(MEs) of the gene module are calculated using the first principal component, representing the overall expression level of the module. By calculating the module-trait correlation coefficient, a heatmap of the correlation coefficient between the module and the trait is generated. Gene modules that are significantly associated with the phenotype are then screened based on the correlation coefficient between the module's eigenvector and the trait, as well as the module significance (P < 0.05). Finally, the correlation coefficients (GS) between gene expression and the trait within a module and the module membership (MM) between the expression of a specific gene and the expression of the principal components of genes within the module are calculated. By setting the range of values ​​for GS, MM, and q.weighted, the gene list calculated by the networkScreening function is filtered to identify and characterize key hub genes.

[0048] 4. Results

[0049] 4.1 WGCNA Network Construction

[0050] First, gene expression values ​​of all samples were clustered and phenotypic heatmaps were analyzed. Using the WGCNA algorithm, based on the scale-free network fitting index and average connectivity, power values ​​with a scale-free topology index ≥ 0.85 and good average connectivity were selected. β = 8 was calculated and selected as the soft threshold for this dataset, which was used for the subsequent calculation of the adjacency matrix and TOM matrix between genes.

[0051] 4.2 Correlation analysis between co-expression modules and clinical phenotypes

[0052] The relationship between each gene module and the clinical phenotype was calculated, and it was found that the RBP1 gene was lowly expressed in atrial fibrillation patient samples.

[0053] II. Target Gene Validation

[0054] Peripheral blood leukocytes were collected from nine patients with atrial fibrillation (AF) at the General Hospital of the People's Liberation Army. The diagnostic criteria for AF were: an AF electrocardiogram recorded by a surface electrocardiogram or a single-lead electrocardiogram recording device, lasting >30 seconds. To reduce potential confounding factors, patients with serious primary diseases such as liver, kidney, or hematopoietic system diseases; patients with malignant tumors; and patients with acute diseases such as myocardial infarction, acute heart failure, or acute infection were excluded. Simultaneously, peripheral blood leukocytes from nine individuals undergoing physical examinations of the same age group at the General Hospital of the People's Liberation Army were collected as controls. The expression level of RBP1 in peripheral blood leukocytes of both AF patients and controls was detected by RT-qPCR.

[0055] Peripheral blood leukocyte extraction: Collect 3-5 ml of venous blood, anticoagulate with EDTA, and centrifuge at 2500 rpm for 10 min; carefully aspirate the supernatant plasma and aliquot it into three 0.5 ml centrifuge tubes; add three times the volume of lysed blood to the blood cells, shake well, and incubate on ice for 15 min; centrifuge at 2500 rpm for 10 min and discard the supernatant; add 10 ml of lysed blood, shake well, and incubate on ice for 15 min; centrifuge at 3000 rpm for 10 min and discard the supernatant; invert the centrifuge tubes to remove residual liquid; obtain leukocytes and store at -80℃, avoiding repeated freeze-thaw cycles; the interval between blood collection and leukocyte separation should not exceed 2 hours at room temperature or 5 hours at 4℃ to prevent leukocyte autolysis.

[0056] RNA extraction was performed. Frozen leukocytes were thawed and resuspended in 1 ml of sterile PBS, then centrifuged at 1000 rpm for 1 min at 4°C. The supernatant was discarded, and 1 ml of TRIzol was added to lyse the cells. The mixture was then pipetted and incubated at room temperature for 5 min. The supernatant was transferred to a 1.5 ml EP tube and centrifuged at 12000 rpm for 5 min. The supernatant was collected, chloroform was added, and the mixture was vortexed and incubated at room temperature for 15 min to allow for natural phase separation. The sample was centrifuged at 12000 rpm for 15 min at 4°C. The sample separated into three layers: a yellow organic layer, and colorless water in the middle and upper layers. The RNA was mainly in the aqueous phase. Carefully aspirate the upper aqueous phase into a new 1.5 ml EP tube, add an equal volume of ice-cold isopropanol, and incubate at -20°C for 1 hour. Centrifuge at 12000 rpm for 10 minutes at 4°C. Discard the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water), gently vortex the EP tube to resuspend the precipitate. Centrifuge at 8000 rpm for 5 minutes at 4°C, discard the supernatant, and air dry at room temperature for 5–10 minutes. Dissolve the RNA precipitate in 50 μl of DEPC water, detect the RNA concentration using a spectrophotometer, and store at -80°C for later use.

[0057] For cDNA synthesis, 4.0 μl of 5× PrimeScript Buffer, 1.0 μg of Total RNA, and 20 μl of RNase-Free ddH2O were gently mixed and reacted at 37°C for 15 min, then at 85°C for 5 s, and gradually cooled to 4°C.

[0058] For RT-qPCR, mix 10.0 μl TBGreenPremixExTaqII, 1.0 μl ForwardPrimer, 1.0 μl ReversePrimer, 2.0 μl cDNA, and 20 μl RNase-FreeddH2O thoroughly. Perform pre-denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s and annealing at 60°C for 30–34 s, for a total of 40 cycles.

[0059] The Ct value of the target gene was normalized by standardizing the Ct value of the internal reference gene GAPDH, and then 2... -△△Ct The expression level of RBP1 was obtained by performing relative quantitative analysis of gene expression differences in the sample.

[0060] like Figure 1 As shown, the expression level of RBP1 in peripheral blood leukocytes of patients with atrial fibrillation was significantly lower than that in controls (P = 0.004). Figure 2 As shown, the diagnostic value of RBP1 for atrial fibrillation was analyzed using ROC curve analysis. The results showed that the area of ​​AUC was 0.8519 and P = 0.0118, indicating that the expression level of RBP1 has high diagnostic value for atrial fibrillation and is expected to become a biomarker for the diagnosis of atrial fibrillation.

[0061] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Use of an agent that detects RBP1 for the manufacture of a kit for the diagnosis of atrial fibrillation, characterized in that: A reagent for detecting RBP1 mRNA. A reagent for detecting RBP1 mRNA.

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