Reference gene for miRNA detection and related products and applications thereof

Abstract

This invention discloses an internal reference gene suitable for miRNA detection, along with related products and applications, relating to the field of bioassay. Through experimental testing and data analysis using different methods on miRNAs in a fever control group and KD patients, this invention discovered that the microRNA miR-30d-5p can serve as a standardized internal reference gene for the detection of platelet microRNAs in Kawasaki disease, exhibiting good stability and accuracy.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more specifically, to internal reference genes suitable for miRNA detection, as well as related products and applications. Background Technology

[0002] Kawasaki disease (KD) is an acute, self-limiting vasculitis, primarily affecting children and constipating as an inflammatory syndrome. It mainly affects small and medium-sized arteries, particularly the coronary arteries, and is a leading acquired disease. KD can occasionally be fatal, especially if missed or left untreated. Clinically, KD presents with prolonged fever and at least four to five of the following features: fever lasting more than five days, limb changes (swelling and peeling of the hands and feet), rash, non-exudative conjunctivitis, oral changes, and cervical lymphadenopathy (usually unilateral). Approximately one-quarter of KD patients present with incomplete clinical manifestations, meaning they lack full major clinical presentations, which delays diagnosis and increases the risk of developing the disease. This results in an incomplete clinical diagnostic standard for Kawasaki disease. Although the reliability of the diagnosis, various novel biomarkers, and classification tools have been investigated, no KD-specific biomarkers have been identified to date.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide internal reference genes suitable for miRNA detection, as well as related products and applications.

[0005] This invention is implemented as follows:

[0006] In a first aspect, embodiments of the present invention provide the application of the miR-30d-5p detection reagent in the preparation of detection reagents for internal reference genes suitable for miRNA detection.

[0007] Secondly, embodiments of the present invention provide the application of miR-30d-5p detection reagent in the preparation of products for detecting Kawasaki disease.

[0008] Thirdly, embodiments of the present invention provide a reagent or kit comprising: a detection reagent for miRNA and a detection reagent for an internal reference gene; wherein the internal reference gene includes miR-30d-5p.

[0009] Fourthly, embodiments of the present invention provide a method for detecting the expression level of a target miRNA in a sample, the method comprising using miR-30d-5p as an internal reference gene to analyze the expression level of the target miRNA.

[0010] The present invention has the following beneficial effects:

[0011] This invention, through experimental testing and data analysis of miRNAs in a fever control group and a KD patient group, discovered that the microRNA miR-30d-5p can be used as a standardized internal reference gene for the detection of platelet microRNAs in Kawasaki disease, and has good stability and accuracy. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 Comparison of the average expression Ct values ​​of the four miRNAs;

[0014] Figure 2 Comparison of the coefficients of variation for the four miRNAs;

[0015] Figure 3 geNorm was used to analyze the expression stability of four miRNA genes;

[0016] Figure 4 NormFinder was used to analyze the expression stability values ​​of four miRNA genes.

[0017] Figure 5 Relative expression of let-7g-5p normalized to miR-126-3p (A) and relative expression of let-7g-5p normalized to miR-30d-5p (B);

[0018] Figure 6 Relative expression of miR-26a-5p normalized to miR-126-3p (A) and relative expression of miR-26a-5p normalized to miR-30d-5p (B);

[0019] Figure 7 ROC curves of let-7g-5p were standardized for miR-126-3p and miR-30d-5p, respectively;

[0020] Figure 8 ROC curves of miR-26a-5p were standardized for miR-126-3p and miR-30d-5p, respectively. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] miRNAs (microRNAs) are a class of endogenous non-coding small RNAs, approximately 22 nucleotides in length, that regulate gene expression at the post-transcriptional level. Increasing research shows that miRNAs play crucial roles in the regulatory mechanisms of various organisms, including developmental timing and host-pathogen interactions, as well as cell growth, differentiation, proliferation, and apoptosis. miRNAs are also associated with the development and progression of many diseases, including cancer, diabetes, heart failure, acute myocardial infarction, allergic inflammatory responses, and tissue damage. Numerous studies have confirmed significant changes in miRNA expression under normal physiological conditions and disease states, demonstrating the enormous potential and broad prospects of miRNAs as non-invasive molecular diagnostic biomarkers in disease diagnosis and prognosis.

[0023] Real-time quantitative PCR (RT-qPCR) has become a commonly used method for miRNA gene expression analysis due to its advantages such as high sensitivity, high specificity, good reproducibility, and high throughput. RT-qPCR is divided into absolute quantification and relative quantification. Relative quantification does not require known amounts of standards, hence its frequent use. However, this method requires data correction of the target gene using an internal reference gene to obtain accurate results. Although some studies have identified and evaluated optimal reference genes for serum miRNA standardization (e.g., patent application number 201210065684.4), internal reference genes stably expressed under certain conditions may vary under other experimental conditions. Internal reference genes that are stably expressed under all experimental conditions are almost nonexistent. Therefore, it is necessary to screen from numerous internal reference genes to select those stably expressed under specific experimental conditions and in specific experimental projects.

[0024] Currently, standardized miRNA detection uses exogenous, artificially synthesized non-human miRNAs (such as those from nematodes) as external references. However, external references can only serve as quality control for the effectiveness of sample extraction and detection; they cannot reflect the expression status of endogenous miRNAs in the sample or differences in sample collection. Commonly used internal reference genes for miRNA detection include 5S rRNA, 18S rRNA, U6, and miRNAs. However, these miRNAs are not identical, do not belong to the same gene family, and may differ from miRNAs in reverse transcription and PCR amplification efficiency. Therefore, standardized miRNA detection is not suitable.

[0025] This invention extracts small RNA molecules less than 200 nt in length from platelet samples of KD and fever control groups, and uses stem-loop reverse transcription primers combined with fluorescent probes (RT-PCR) with higher specificity and sensitivity to analyze differentially expressed miRNAs. Through a series of data analyses, the platelet miRNA internal reference gene miR-30d-5p was screened out. Compared with other internal reference genes (such as miR-126-3p, The Platelet microRNA Profile of Kawasaki Disease: Identification of Novel Diagnostic Biomarkers), miR-30d-5p has better stability and higher expression level in the detection of platelet miRNAs, and has higher accuracy and discriminative power for the detection of differentially expressed miRNAs in KD group and fever control group samples.

[0026] Specifically, this invention provides the application of miR-30d-5p detection reagent in the preparation of detection reagents for internal reference genes suitable for miRNA detection.

[0027] In some embodiments, the detection reagent for miR-30d-5p includes at least one of primer pairs, probes, and chips for detecting miR-30d-5p.

[0028] Understandably, the specific sequences of primer pairs and probes for detecting miR-30d-5p can be obtained based on conventional primer and probe design methods. The contribution of this application is that it discovers that using miR-30d-5p as an internal reference gene for detecting the expression level of miRNA markers in Kawasaki disease can improve the stability and effectiveness of Kawasaki disease detection.

[0029] In some embodiments, the primer pair for detecting miR-30d-5p includes: a reverse transcription primer and / or a detection primer pair. The nucleotide sequence of the reverse transcription primer may be as shown in SEQ ID NO:1, the nucleotide sequence of the detection primer pair may be as shown in SEQ ID NO:2-3, and the nucleotide sequence of the probe may be as shown in SEQ ID NO:4.

[0030] In some embodiments, the miRNA includes platelet miRNA.

[0031] In some embodiments, the miRNA includes a miRNA biomarker for detecting Kawasaki disease.

[0032] On the other hand, embodiments of the present invention provide the application of miR-30d-5p detection reagent in the preparation of products for detecting Kawasaki disease.

[0033] In some embodiments, the product is selected from any one of reagents, kits, chips, and prediction models.

[0034] In some embodiments, the detection reagent for miR-30d-5p can be the same as that described in any of the foregoing embodiments.

[0035] On the other hand, embodiments of the present invention provide a reagent or kit comprising: a detection reagent for miRNA and a detection reagent for an internal reference gene; wherein the internal reference gene comprises miR-30d-5p.

[0036] In some embodiments, the miRNA detection reagent includes a detection reagent for detecting miRNA biomarkers of Kawasaki disease.

[0037] In some embodiments, the miRNA biomarkers for detecting Kawasaki disease include let-7g-5p and miR-26a-5p.

[0038] In some embodiments, the detection reagent for let-7g-5p includes at least one of a primer pair, a probe, and a chip for detecting let-7g-5p. Optionally, the primer pair for detecting let-7g-5p includes a reverse transcription primer and / or a detection primer pair. The nucleotide sequence of the reverse transcription primer may be as shown in SEQ ID NO:9, the nucleotide sequence of the detection primer pair may be as shown in SEQ ID NO:10-11, and the nucleotide sequence of the probe may be as shown in SEQ ID NO:12.

[0039] In some embodiments, the miR-26a-5p detection reagent includes at least one of a primer pair, a probe, and a chip for detecting miR-26a-5p. Optionally, the primer pair for detecting miR-26a-5p includes a reverse transcription primer and / or a detection primer pair. The nucleotide sequence of the reverse transcription primer may be as shown in SEQ ID NO:13, the nucleotide sequence of the detection primer pair may be as shown in SEQ ID NO:14-15, and the nucleotide sequence of the probe may be as shown in SEQ ID NO:16.

[0040] In some embodiments, the detection reagent for the internal reference gene includes at least one of a primer pair, a probe, and a chip for detecting miR-30d-5p.

[0041] Furthermore, embodiments of the present invention provide a method for detecting the expression level of a target miRNA in a sample, the method comprising using miR-30d-5p as an internal reference gene to analyze the expression level of the target miRNA.

[0042] In some embodiments, the target miRNA includes: a miRNA biomarker for detecting Kawasaki disease.

[0043] In some embodiments, the miRNA markers include: let-7g-5p and miR-26a-5p;

[0044] In some embodiments, the method includes: obtaining a cDNA template after reverse transcription of platelet miRNA from a sample; performing quantitative real-time PCR amplification of the cDNA template using primers for detecting the target miRNA and the internal reference gene; and obtaining the expression level of the target miRNA by comparing the Ct values ​​of the internal reference gene and the target miRNA.

[0045] In some embodiments, the method is not directly aimed at diagnosing or treating a disease.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] The sample collection process and validation experiments have been approved by the hospital's ethics committee.

[0048] The relevant detection primer and probe sequences used in the examples are as follows:

[0049] The reverse transcription primers for miR-30d-5p detection and the primers and probes for real-time PCR detection are as follows.

[0050]

[0051] The reverse transcription primers for miR-126-3p detection and the primers and probes for quantitative real-time PCR detection are as follows.

[0052]

[0053] The sequences of the reverse transcription primers for let-7g-5p detection and the primers and probes for quantitative real-time PCR detection are as follows.

[0054]

[0055] The reverse transcription primers for miR-26a-5p detection and the primers and probes for quantitative real-time PCR detection are as follows.

[0056]

[0057] Example 1

[0058] 1. Sample and platelet RNA extraction

[0059] Peripheral blood samples (2.0 ml) from children with kappa, dysplasia, and fever were collected using EDTA (ethylenediaminetetraacetic acid) anticoagulant tubes. The peripheral blood was centrifuged at 120g for 20 min at 4°C, and the supernatant (pale yellow liquid) was transferred to a 2.0 ml nuclease-free centrifuge tube. The tube was then centrifuged at 360g for 20 min at 4°C, and the supernatant was discarded. The platelet pellet was washed twice with 1×PBS, and the PBS supernatant was discarded after washing. miRNA was extracted from platelets according to the miRNA extraction kit instructions. The concentration and purity of platelet miRNA were determined using Qubit and Nanodrop. Qualified samples can be stored long-term at -80°C or used directly for subsequent operations.

[0060] 2. Detection of miRNA reverse transcription and real-time quantitative PCR

[0061] All clinical kDay disease (KD) samples and fever control samples (8 μl per sample) were reverse transcribed into cDNA using a miRNA reverse transcription kit and stem-loop reverse transcription primers. PCR detection was then performed using PCR Mix, appropriate primers, probes, and a quantitative real-time PCR instrument (ABI 7500). All samples were tested three times, and the threshold values ​​for the detection results were set to a fixed value. The reverse transcription and PCR detection systems are as follows.

[0062] (1) removal of gDNA (genomic DNA)

[0063] Table 1 Reaction System

[0064] Components Volume (μl) DNAMix 2 miRNA 8 total 10

[0065] After the reaction system is prepared and mixed by blowing, it is incubated at 42°C for 2 minutes.

[0066] (2) Reverse transcription and cDNA synthesis

[0067] Table 2 Reaction System

[0068]

[0069]

[0070] After the reaction solution is prepared and mixed, cDNA synthesis is carried out under the following conditions.

[0071] Table 3 Reaction conditions

[0072] reaction temperature time 25℃ 5min 50℃ 15min 85℃ 5min

[0073] The PCR reaction solution for reverse-transcribed cDNA was prepared according to the following system.

[0074] Table 4 Reaction System

[0075] Components volume Probe qPCR Mix (2x) 10μl Primer-F (10μm) 0.4μl Primer-R (10μm) 0.4μl Probe (10μm) 0.2μl Template cDNA 1μl RNase-free water 7.6μl 50×ROX Reference Dye 2 0.4μl Total volume 20μl

[0076] The PCR reaction procedure is as follows.

[0077] Table 5 Reaction Procedure

[0078]

[0079] 3. Results Analysis - Expression Pattern Analysis of Different Candidate Genes

[0080] RT-PCR detection results of four miRNAs were performed on 42 samples (see Table 6) from 14 Kawasaki disease patients before treatment, 10 Kawasaki disease patients after treatment, and 18 febrile controls. A fixed threshold was set, and each sample was tested three times. The Ct values ​​and coefficients of variation of each candidate gene were analyzed. The results are shown in Table 6. Figure 1 and Figure 2 The expression levels of different miRNAs varied considerably, with Ct values ​​ranging from 24.64 to 30.3. Among them, miR-30d-5p had the highest expression level (Ct value of 24.64), while let-7g-5p had the lowest expression level (Ct value of 30.3). miR-30d-5p also had the lowest coefficient of variation, approximately 0.063. Considering the selection criteria of high expression level (low Ct value) and low coefficient of variation, miR-30d-5p is more suitable as a standardized internal reference gene.

[0081] Table 6 Sample Information

[0082]

[0083]

[0084] 4. Gene stability analysis

[0085] The expression stability of candidate reference genes was calculated using geNorm and NormFinder software, respectively.

[0086] ① The geNorm software is specifically designed for screening and determining the number of internal reference genes in real-time quantitative PCR. This program can screen any number of internal reference genes for any experiment and ultimately select two or more gene combinations to correct the data, resulting in more accurate relative quantification. The program uses the HK function algorithm to sort the selected reference / housekeeping genes, calculating the gene stability measure M of all candidate genes and excluding genes with the highest M value. Then, it calculates the gene stability measure M of the remaining genes, and so on. This process is repeated until two minNrHK genes are retained, with the smaller the M value, the more stable the gene. The software can also calculate the paired variance V of the normalized factor after introducing a new internal reference gene and determine the optimal number of internal reference genes based on the Vn / Vn+1 value. The default V value is 0.15 (this value can be adjusted slightly manually). If the Vn / Vn+1 value is less than 0.15, the optimal number of internal reference genes is n; if the Vn / Vn+1 value is greater than 0.15, the optimal number of internal reference genes is n+1.

[0087] The stability analysis results of candidate genes in all clinical samples (KD patients and fever control group) using geNorm software are as follows ( Figure 3 Based on the average expression stability values ​​(M) in samples before and after KD treatment and in the fever control group, miR-30d-5p showed the best stability, followed by miR-126-3p and let-7g-5p. This means that miR-30d-5p had the lowest stability value and the best stability. Furthermore, the stability of miR-30d-5p (M value 0.81) was better than that of miR-126-3p (M value 0.86), indicating that miR-30d-5p is more suitable as an internal control.

[0088] ②The NormFinder program is an algorithm that identifies the optimal normalized gene from a set of candidate genes. Based on a mathematical model of gene expression, this algorithm uses a reliable statistical framework to estimate not only the overall expression changes of candidate normalized genes but also to calculate the differences between different sample sets, such as normal and cancer samples. The NormFinder program provides a stability value for each gene. Its calculation principle is similar to the geNorm program: it first obtains the stable expression values ​​of candidate reference genes, and then selects the most suitable reference gene based on the magnitude of the stability value. The criterion for selection is that the reference gene with the smallest expression value M is the most stable reference gene.

[0089] The results of the NormFinder program's analysis of candidate gene stability for all clinical samples (KD group and fever control group) are as follows ( Figure 4The stability of miR-30d-5p was the lowest (0.131), indicating the best stability, followed by miR-126-3p (0.167). This result is consistent with the results of the geNorm program, and both internal reference screening software results show that miR-30d-5p is more stable than miR-126-3p. Combining the analysis results of the two software programs, miR-30d-5p has better expression stability, indicating that miR-30d-5p is more suitable as an internal reference.

[0090] 5. Comparison of the effects of calibration of different internal reference genes on miRNA quantification results

[0091] Previous studies have confirmed that let-7g-5p and miR-26a-5p showed significant differences in expression in platelet samples from children with KD and the fever control group, and that the expression of these two miRNAs was upregulated in platelets from children with KD.

[0092] In this embodiment, quantitative real-time PCR was used to detect two miRNAs in platelets of children with kinetinopathy (KD) and a febrile control group. The relative expression of miRNAs was analyzed using the 2-ΔΔCT method. The candidate internal reference genes miR-126-3p and miR-30d-5p were used as internal reference genes for standardization calibration. The results were analyzed for statistical significance using the t-test. A p-value less than 0.05 was considered statistically significant, and the smaller the p-value, the more significant the difference, indicating a more significant difference in the expression levels of let-7g-5p and miR-26a-5p in platelets of children with KD.

[0093] The results show that... Figure 5 and 6 The expression of let-7g-5p calibrated with miR-126-3p was statistically significant in the fever control group and before KD treatment (KDSB) (significant difference, P value 0.02), and its expression level was upregulated before KD treatment.

[0094] The expression levels of let-7g-5p calibrated using miR-30d-5p were statistically significant in the fever control group and before KD treatment (KDSB) (significant difference, P value 0.0043). Compared with the results calibrated using miR-126-3p, the calibrated miR-30d-5p showed a smaller P value, indicating that the difference in let-7g-5p between the two groups was more significant, and the trend of upregulation of let-7g-5p expression was more significant in children before KD treatment (KDSB).

[0095] The expression of miR-26a-5p calibrated with miR-30d-5p (P value 5.4e-05) was significantly different from that of miR-26a-5p calibrated with miR-126-3p (P value 3.9e-05) in the febrile control group and before KD treatment (KDSB), indicating that miR-26a-5p had a stronger ability to distinguish between KD cases and febrile patients.

[0096] Example 2

[0097] 2. miR-30d-5p, as an internal reference gene for detecting the expression level of miRNA biomarkers in Kawasaki disease, can improve the stability and effectiveness of Kawasaki disease detection. The stability and effectiveness here are compared using the area under the receiver operating characteristic (AUC) of the normalized ROC curves (ROC curves) of two KD-related biomarkers, let-7g-5p and miR-26a-5p, respectively, with the internal references miR-126-3p and miR-30d-5p. (A larger AUC indicates better classifier performance and higher predictive value.)

[0098] From the ROC curve ( Figures 7-8 It can be seen that the AUC areas of let-7g-5p and miR-26a-5p after being normalized with miR-30d-5p are larger than the AUC areas of miR-126-3p after normalization.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of miR-30d-5p detection reagent in the preparation of detection reagents for internal reference genes suitable for miRNA detection, wherein the miRNA is: a miRNA biomarker for detecting Kawasaki disease.

2. The application according to claim 1, characterized in that, The detection reagent for miR-30d-5p includes at least one of primer pairs, probes, and chips for detecting miR-30d-5p.

3. Application of miR-30d-5p detection reagent in the preparation of products for detecting Kawasaki disease.

4. The application according to claim 3, characterized in that, The product is selected from any one of the following: reagents, kits, chips, and prediction models.

5. The application according to claim 3 or 4, characterized in that, The detection reagent for miR-30d-5p includes at least one of primer pairs, probes, and chips for detecting miR-30d-5p.

6. A reagent or kit, characterized in that, It includes: The detection reagents for miRNA and the detection reagents for internal reference genes; the internal reference gene includes miR-30d-5p; The miRNA detection reagent is a detection reagent for detecting miRNA biomarkers of Kawasaki disease.

7. The reagent or kit according to claim 6, characterized in that, The miRNA biomarkers used to detect Kawasaki disease include: let-7g-5p and miR-26a-5p.

8. The reagent or kit according to claim 6 or 7, characterized in that, The detection reagents for the internal reference gene include at least one of a primer pair, a probe, and a chip for detecting miR-30d-5p.

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