PCR internal reference and preparation method of serum small extracellular vesicle miRNA

Abstract

The present invention proposes a PCR internal reference and preparation method of serum small extracellular vesicle miRNA, which belongs to the field of genetic engineering technology. The internal reference is selected from any one of miR‑30e‑5p, miR‑361‑3p, miR‑93‑5p, miR‑181a‑5p, miR‑425‑5p and miR‑186‑5p or a combination thereof; the nucleotide sequence of miR‑30e‑5p is shown in SEQ ID NO.1. The present invention quantifies the expression of serum small extracellular vesicle miRNA in patients with benign and malignant ovarian tumors, screens the most stable miRNA expressed between different individuals as a candidate internal reference or internal reference combination, and uses real-time fluorescence quantitative PCR technology to verify the stability of the screened candidate internal reference / internal reference combination in another independent ovarian tumor patient population.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a PCR internal reference of serum small extracellular vesicle miRNA and a preparation method thereof. Background Art

[0002] Ovarian cancer is the most deadly gynecological malignancy and the fifth leading cause of cancer-related death in women. Epithelial ovarian cancer is the main type of ovarian cancer, accounting for about 70% of ovarian malignancies. Ovarian cancer has a special anatomical location, deep in the pelvic cavity, and has no obvious early symptoms. Once discovered, it is often in the late stage, with a 5-year survival rate of only 20%-30%. The 5-year survival rate of patients with early ovarian cancer exceeds 90%. Therefore, establishing an effective screening mechanism and striving for early diagnosis of ovarian cancer are of great significance to the treatment and prognosis of patients.

[0003] As an important form of liquid biopsy, free exosomes in peripheral blood have received extensive attention and in-depth research in the fields of early diagnosis of cancer, tumor prognosis monitoring, and immunotherapy. Exosomes exist in almost all biological fluids and contain rich proteins, lipids, mRNA, lncRNA, and miRNA. In recent years, many studies have shown that exosomes secreted by tumor cells have specific overexpressed miRNAs, which may be related to tumor growth and metastasis. At present, a variety of exosomal miRNAs have been found to be associated with ovarian cancer. In an ovarian cancer exosome microarray study, YOSHIMURA et al. found that compared with patients with benign tumors and healthy volunteers, the serum miR-99a-5p level of ovarian cancer patients was significantly increased (1.7 times and 2.8 times, respectively), indicating that miR-99a-5p can be used as a potential marker for early diagnosis of ovarian cancer (PMID: 30396333). Chi used microRNA array and TaqMan real-time PCR technology to analyze the expression levels of plasma exosome miRNA in patients with epithelial ovarian cancer and ovarian cystadenoma and healthy women, and found that compared with healthy women, some miRNAs in patients with epithelial ovarian cancer and ovarian cystadenoma were upregulated or downregulated. Further analysis locked exosomal miR-200b for epithelial ovarian cancer patients with diagnostic and prognostic relevance (PMID: 30107086). Multiple research results show that the expression levels of some exosomal miRNAs may be different in patients with malignant tumors compared with healthy people. Therefore, accurately quantifying the expression levels of miRNAs in exosomes of different individuals is extremely important for improving the accuracy and reliability of biomarker research.

[0004] Real-time fluorescence quantitative PCR technology is an effective means to detect miRNA expression levels. However, there are large differences in RNA extraction quality, extraction yield and reverse transcription efficiency among different samples, which will affect the reliability of the expression difference results of the target gene. The amplification of the internal reference can reflect the sample quality and the stability of the experimental process. When detecting the target gene, selecting a suitable internal reference to correct the results is of great significance to improve the reliability of the quantitative results. However, at present, there is no unified internal reference for exosomal miRNA quantification that is suitable for all sample types. Most studies use classic internal references such as U6, but in fact, U6 is only located in the nucleus and not in the cytoplasm when RNA is loaded into the exosomes. Therefore, the detection of U6 expression may be due to the insufficient purity of the exosome isolation method. In addition, some other commonly used internal references such as miR16 and miR451 have been shown to be enriched in red blood cells and are easily interfered by hemolysis. Some potential internal references for exosomal miRNA have also been reported, but their expression is not stable under different physiological and pathological conditions. Blindly using unverified internal references may lead to wrong or even opposite conclusions. The external reference method is another option for the quantitative analysis of exosome miRNA RT-qPCR. After exosome separation and before RNA extraction, an equal amount of exogenous miRNA is added to correct the deviations in the experimental process of different samples. However, this type of external reference is mainly used to correct the RNA extraction, reverse transcription and PCR process, and cannot correct the most complex exosome separation and enrichment process. Therefore, the external references used in the current exosome miRNA quantitative research cannot fully meet the needs of exosome miRNA RT-qPCR quantitative analysis.

[0005] Due to the difficulty in standardization in the quantitative research of exosomal miRNA, on the one hand, the sequencing data is poorly consistent with the RT-qPCR results, which reduces the possibility of discovering biomarkers. On the other hand, it also limits the application of exosomal miRNA biomarkers from scientific research to clinical transformation.

[0006] In Chinese patent application CN109536502A, researchers found a stably expressed miRNA in plasma exosomes of patients with gestational trophoblastic tumor based on a small number of samples, and used this as a candidate internal reference for verification in another cohort, thereby identifying an internal reference suitable for quantitative analysis of plasma exosome miRNA RT-qPCR in patients with gestational trophoblastic tumor. However, this study found that the sample size of the cohort was too small, and the analysis results were difficult to represent the entire gestational trophoblastic tumor patient population, and the internal reference identified in this study could not be directly used for RT-qPCR quantification of serum small extracellular vesicle miRNA in patients with ovarian tumors. Summary of the invention

[0007] The present invention aims to propose and verify an internal reference composition for RT-qPCR quantification of exosomal miRNA, which can be used to correct the deviations in the experimental process between different samples, better standardize the quantitative data, and improve the accuracy and reliability of exosomal miRNA as a biomarker for the diagnosis or treatment of ovarian tumors.

[0008] The technical solution of the present invention is achieved in this way:

[0009] The present invention provides a PCR internal reference of serum small extracellular vesicle miRNA, wherein the internal reference is selected from any one of miR-30e-5p, miR-361-3p, miR-93-5p, miR-181a-5p, miR-425-5p and miR-186-5p or a combination thereof; the nucleotide sequence of the miR-30e-5p is shown in SEQ ID NO.1; the nucleotide sequence of the miR-361-3p is shown in SEQ ID NO.2; the nucleotide sequence of the miR-93-5p is shown in SEQ ID NO.3; the nucleotide sequence of the miR-181a-5p is shown in SEQ ID NO.4; the nucleotide sequence of the miR-425-5p is shown in SEQ ID NO.5; and the nucleotide sequence of the miR-186-5p is shown in SEQ ID NO.6.

[0010] As a further improvement of the present invention, the internal reference is selected from any one of miR-30e-5p, miR-186-5p, miR-93-5p or a combination thereof.

[0011] As a further improvement of the present invention, the internal reference is a combination of miR-30e-5p and miR-186-5p.

[0012] As a further improvement of the present invention, the internal reference is suitable for RT-qPCR quantification of serum small extracellular vesicle miRNA in patients with ovarian tumors.

[0013] The present invention further protects the use of the above-mentioned internal reference gene in the preparation of a PCR detection kit for serum small extracellular vesicle miRNA or a biomarker or kit for diagnosing ovarian tumors.

[0014] The present invention further protects a method for preparing the above-mentioned serum small extracellular vesicle miRNA PCR internal reference, comprising the following steps:

[0015] S1: extracting serum small extracellular vesicles from serum samples; wherein the serum samples are from samples of patients with benign and malignant ovarian tumors;

[0016] S2: Extraction and expression detection of miRNA in the extracted serum small extracellular vesicles;

[0017] S3: Sequencing, annotation, filtering, and expression level normalization are performed on the extracted miRNA in sequence to obtain the standardized miRNA expression level;

[0018] S4: Screen miRNAs according to the screening criteria and select stable miRNAs as candidate internal references to be verified;

[0019] S5: Reverse transcription and RT-qPCR detection of candidate internal control miRNA to be verified;

[0020] S6: Perform stability verification on the candidate internal references to be verified, and select the best final internal references.

[0021] As a further improvement of the present invention, the method for extracting serum small extracellular vesicles in step S1 includes: at least one of a reagent extraction method, an ultracentrifugation extraction method, a density gradient centrifugation extraction method, an ultrafiltration centrifugation extraction method, and a magnetic bead immunoextraction method.

[0022] As a further improvement of the present invention, the specific method for extracting serum small extracellular vesicle miRNA in step S2 is: using a miRNeasy Serum / Plasma Kit to extract serum small extracellular vesicle miRNA.

[0023] As a further improvement of the present invention, the screening criteria in step S4 are:

[0024] 1) The expression level is high in patients with benign and malignant ovarian tumors, and the normalized expression value is greater than 8;

[0025] 2) The coefficient of variation is small and the expression is stable.

[0026] As a further improvement of the present invention, step S4 specifically includes:

[0027] S41: Eliminate miRNAs with expression levels less than 8 after normalization;

[0028] S42: Calculate the coefficient of variation of each remaining miRNA and sort the coefficients of variation from small to large;

[0029] S43: Selecting miRNAs ranked in the top N in terms of coefficient of variation, and performing stability analysis on the expression levels of the selected miRNAs;

[0030] S44: Select the top M miRNAs with the highest comprehensive stability as candidate internal references to be verified;

[0031] Wherein, N and M are natural numbers, and N is greater than M.

[0032] As a further improvement of the present invention, the value of N is 60, and the value of M is 6.

[0033] As a further improvement of the present invention, the candidate internal references to be verified include miR-30e-5p, miR-361-3p, miR-93-5p, miR-181a-5p, miR-425-5p and miR-186-5p.

[0034] As a further improvement of the present invention, the method for performing stability verification on the candidate internal reference to be verified in step S6 includes:

[0035] S61: taking the candidate internal reference to be verified and miR-451a which is frequently used as an internal reference as the internal reference to be analyzed;

[0036] S62: performing RT-qPCR detection on the internal reference to be analyzed in serum small extracellular vesicle samples of several patients with benign ovarian lesions and several patients with malignant ovarian tumors; wherein the expression level of the internal reference to be analyzed is represented by a Ct value;

[0037] S63: Evaluate the stability of the Ct value of the internal reference to be analyzed, and verify the internal reference to be analyzed based on the stability evaluation.

[0038] The present invention further protects a detection reagent, including the above-mentioned PCR internal reference suitable for serum small extracellular vesicle miRNA of ovarian tumor patients.

[0039] The present invention further protects a detection kit, comprising the above detection reagent.

[0040] The present invention further protects the use of the above detection kit in RT-qPCR quantification of ovarian tumors.

[0041] The present invention has the following beneficial effects:

[0042] (1) The present invention discovered and published for the first time six potential internal reference miR-30e-5p, miR-361-3p, miR-93-5p, miR-181a-5p, miR-425-5p and miR-186-5p that can be used for RT-qPCR quantification of serum small extracellular vesicle miRNA in patients with ovarian tumors.

[0043] (2) The internal reference miRNA proposed in the present invention is an endogenous molecule in exosomes and is more reliable than the intracellular nuclear small RNA (snRNA) U6. In addition, the internal reference is stably expressed and has a high abundance among different individuals of ovarian tumor patients. It has been verified by different cohort samples and has a high credibility. It can be used to correct the deviation in the experimental process between different samples, better standardize the quantitative data, and improve the accuracy and reliability of exosome miRNA as a biomarker for tumor diagnosis or treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0045] Figure 1 Transmission electron microscopy identification results of small extracellular vesicles;

[0046] Figure 2 The results are the expression results of proteins characteristic of small extracellular vesicles;

[0047] Figure 3 This is the statistical result diagram of the Ct value distribution of the validation cohort of the small extracellular vesicle miRNA candidate reference gene miR-30e-5p;

[0048] Figure 4 This is the statistical result diagram of the Ct value distribution of the validation cohort of the small extracellular vesicle miRNA candidate reference gene miR-181a-5p;

[0049] Figure 5 This is the statistical result diagram of the Ct value distribution of the validation cohort of the small extracellular vesicle miRNA candidate reference gene miR-361-3p;

[0050] Figure 6 This is the statistical result diagram of the Ct value distribution of the validation cohort of the small extracellular vesicle miRNA candidate reference gene miR-93-5p;

[0051] Figure 7 This is the statistical result diagram of the Ct value distribution of the validation cohort of the small extracellular vesicle miRNA candidate reference gene miR-425-5p;

[0052] Figure 8 This is the statistical result diagram of the Ct value distribution of the validation cohort of the small extracellular vesicle miRNA candidate reference gene miR-186-5p;

[0053] Fig. 9This is the statistical result diagram of the Ct value distribution of the validation cohort of the small extracellular vesicle miRNA candidate reference gene miR-451a;

[0054] Fig.10 This is a comparison chart of comprehensive gene stability;

[0055] Fig.11 This is a comparison chart of gene stability analyzed using the △Ct method;

[0056] Fig.12 This is a comparison chart of gene stability analyzed using BestKeeper software;

[0057] Fig.13 This is a comparison chart of gene stability analyzed using normFinder software;

[0058] Fig.14 This is a comparison chart of gene stability analyzed using Genorm software. DETAILED DESCRIPTION

[0059] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] Example 1

[0061] This embodiment provides a PCR internal reference suitable for serum small extracellular vesicle miRNA of ovarian tumor patients, and the preparation method is as follows:

[0062] (1) Serum sample collection

[0063] This study included 135 samples of patients with benign and malignant ovarian tumors in the training cohort and validation cohort. Blood samples were collected in 10ml vacuum blood collection tubes (REF367820, BD, USA) before drug treatment. The tubes were slowly and gently turned upside down several times, placed vertically, and left at room temperature for 1-2 hours. After the blood clots solidified, they were first centrifuged at room temperature at 2000g for 10 minutes. Samples with hemolysis levels less than 4 were used for subsequent research. After centrifugation, the supernatant was transferred to a new 1.5ml centrifuge tube and centrifuged again at 8000g and 4 degrees for 10 minutes. After centrifugation, 1ml of the supernatant was transferred to a new 1.5ml centrifuge tube, and the serum was stored in a refrigerator at minus 80 degrees.

[0064] (2) Extraction and characterization of serum small extracellular vesicles

[0065] 1) Extraction of serum small extracellular vesicles

[0066] The extraction of serum microvesicles was carried out using the L-type exosome precipitation agent independently developed by 3DMed (L3525, 3DMed, Shanghai, China). The specific steps are as follows: ① Take out the frozen serum sample and incubate it at 37°C until it is completely thawed, and centrifuge it at 12000g and 4°C for 10min; ② Transfer the supernatant to a 0.45μm tube filter (Costar, CLS8163-100EA, Corning, USA), centrifuge it at 12000g and 4°C for 5min, and filter 500ul each time until all samples are completely filtered; ③ Transfer the filtrate to a 0.22μm tube filter (Costar, CLS8161-100EA, USA) 12000g, centrifuge at 4℃ for 5min, filter 500ul each time until all samples are completely filtered; ④ Transfer the filtrate to a 1.5ml centrifuge tube and add 1 / 4 volume of L3525; ⑤ Vortex to mix, incubate at 4℃ for 30min; ⑥ 4700g, centrifuge at 4℃ for 30min; ⑦ After centrifugation, discard the supernatant and add 200μl PBS (phosphate buffer saline) to thoroughly resuspend the small extracellular vesicle precipitate.

[0067] 2) Characteristics of serum small extracellular vesicles

[0068] In order to detect the characteristics of serum small extracellular vesicles in patients with benign ovarian lesions and malignant tumors, the present application uses a transmission electron microscope to detect the morphology of small extracellular vesicles, and uses a fully automatic exosome fluorescence detection and analysis system to detect the expression level of characteristic proteins of small extracellular vesicles. Identification of morphological characteristics of serum small extracellular vesicles: First, resuspend the small extracellular vesicles in PBS, add 4% paraformaldehyde to fix the small extracellular vesicles, and then transfer them to a carbon-coated electron microscope copper grid. After washing the copper grid with PBS twice, wash the copper grid with PBS containing glycine (50mM) for 3min, then wash the copper grid with PBS containing 0.5% BSA for 10min, and finally stain the copper grid with 2% uranyl acetate. After staining, use a transmission electron microscope (H-7650, Hitachi High-Technologies, Japan) to observe the morphological characteristics of small extracellular vesicles.

[0069] Detection of characteristic proteins of small extracellular vesicles: Dilute the isolated small extracellular vesicles to an appropriate concentration, about 1*10^7-1*10^8 / ml, pipette 50ul of the diluted sample and drop it on the chip, and incubate it at room temperature overnight. After washing, add premixed anti-CD9 ( 488A), anti-CD81( 555) and anti-CD63( 647) stain for immunofluorescence staining, wash the antibody again and dry the chip, put the dried chip into the card slot, and finally use the fully automatic exosome fluorescence detection and analysis system (ExoView R100, NanoView Biosciences, USA) for detection.

[0070] (3) Extraction and expression detection of serum small extracellular vesicle miRNA

[0071] 1) Extraction of serum small extracellular vesicle miRNA

[0072] The serum extracellular vesicle miRNA was extracted using miRNeasy Serum / Plasma Kit (217184, QIAGEN, Shanghai, China). The specific operation procedures were referred to the product manual. The miRNA quantification and fragment distribution were detected using the chip of Agilent 2100 Bioanalyzer (5067-1548, Agilent, USA).

[0073] 2) Detection of serum small extracellular vesicle miRNA expression

[0074] This application uses the NEBNext, Multiplex Small RNA Library Prep Set for Illumina (E7300L, NEB, USA) kit for library construction, and small RNA sequencing is used to detect the expression level of serum small extracellular vesicle miRNA. The operation process refers to the product manual. The general process is that the sample loading amount for each sample library construction is 100 ng, the volume does not exceed 6 μl, and 3' adapter connection, reverse transcription primer hybridization, 5' adapter connection, reverse transcription and PCR amplification are performed respectively. Finally, the PCR product is purified using the NucleoSpin Gel and PCR Clean-up (740609.50, MACHEREY-NAGEL, Germany) kit, and the library DNA is eluted with 30 μl of enzyme-free water. GX Touch TM The library quality was checked using the HT nucleic acid analyzer and its supporting chips (CLS138948, PerkinElmer, USA) and reagents (CLS760672, PerkinElmer, USA). 20-25 libraries were mixed in equal molar ratios for lane sequencing. The sequencing platform and mode were Illumina HiSeq PE150.

[0075] (4) Sequencing data analysis process

[0076] 1) Sequencing data alignment. After removing the sequencing adapters of the small RNA sequencing data, the sequencing data were aligned to the human reference genome hg19 (genome download link: http: / / hgdownload.soe.ucsc.edu / goldenPath / hg19 / bigZips / ) using BWA software (version: 0.7.12-r1039), and the number of reads aligned to miRNA was counted.

[0077] 2) miRNA annotation: miRNAs were annotated using the Gencodev25 and miRBasev21 databases, and those annotated as known mature miRNAs were retained for subsequent analysis.

[0078] 3) miRNA filtering: Mature miRNAs with a length of less than or equal to 30 nt and covered by at least 2 reads per sample were retained for subsequent analysis.

[0079] 4) Standardization of miRNA expression: The trimmed mean of M-values ​​(TMM) and limma-voom methods in the limma analysis package in R language were used to standardize the miRNA expression of the training cohort samples.

[0080] (5) Screening of candidate internal references

[0081] Based on small RNA sequencing detection technology, the expression level of miRNA in the patient's peripheral blood extracellular vesicles was obtained, and candidate internal references were screened according to the following principles:

[0082] 1) The expression level is high in patients with benign and malignant ovarian tumors, and the normalized expression level is required to be greater than 8;

[0083] 2) Statistical analysis was performed based on the coefficient of variation CV value (= standard deviation / mean) of miRNA expression, and the top 60 miRNAs with the highest CV value were selected for stability analysis. The gene stability analysis method was a comprehensive online analysis and evaluation software RefFinder, which integrated four internal reference stability analysis methods: geNorm (Vandesompele et al., 2002), NormFinder (Andersen et al., 2004), BestKeeper (Pfaffl et al., 2004) and Delta Ct (Silver et al., 2006).

[0084] (6) Serum small extracellular vesicle miRNA reverse transcription and RT-qPCR detection

[0085] TaqMan advanced miRNA cDNA synthesis kit (A28007, Applied Biosystems TM ) and TaqMan TM Fast Advanced Master Mix (4444557, Applied Biosystems TM ) Perform miRNA reverse transcription and qPCR reaction on 32 samples of the validation cohort. The specific steps are as follows:

[0086] 1) Prepare the following poly(A) tailing reaction mixture in a 1.5 mL centrifuge tube:

[0087] Composition 1 reaction 10×Poly(A)Buffer 0.5μL ATP 0.5μL Poly(A)Enzyme 0.3μL RNase-free water 1.7μL Total volume 3μL

[0088] 2) Take new reaction tubes, add 2 μL of sample and 3 μL of mixed solution to each reaction tube, and the total volume of each reaction tube is 5 μL. Vortex thoroughly to mix, centrifuge all components to the bottom of the tube, place the reaction tube on the PCR instrument, and run according to the following program:

[0089]

[0090]

[0091] 3) After the tailing reaction is completed, the subsequent ligation reaction needs to be carried out immediately. In a 1.5 mL centrifuge tube, prepare the following ligation reaction premix according to the number of reactions.

[0092] Components 1 reaction 5×DNA Ligase Buffer 3μL 50% PEG8000 4.5μL 25×Ligase Adaptor 0.6μL RNA Ligase 1.5μL RNase-free water 0.4μL Overall system 10μL

[0093] 4) Add 10 μL of the ligation reaction system to the reaction tube containing the poly(A) tailing reaction product (from the first step of tailing reaction), with a total reaction volume of 15 μL per tube. Vortex briefly to mix and centrifuge. Place the reaction tube on the PCR instrument and set the reaction program:

[0094] step temperature Duration connect 16℃ 60 minutes Keep 4℃ At least 5 minutes

[0095] 5) After the ligation reaction is completed, reverse transcription reaction is immediately carried out. In a 1.5 mL centrifuge tube, prepare the following reverse transcription mixture according to the number of reactions:

[0096]

[0097]

[0098] 6) Add 15 μL of reverse transcription mixture to the reaction tube containing the ligation reaction product (total reaction volume per tube is 30 μL), vortex briefly to mix, centrifuge, place the reaction tube on the PCR instrument, and set the reaction program:

[0099] step temperature Duration Reverse transcription reaction 42℃ 15 minutes Stop the reaction 85℃ 5 minutes Keep 4℃ At least 5 minutes

[0100] 7) After the reverse transcription reaction, perform miRNA pre-amplification. In a 1.5 mL centrifuge tube, prepare the following pre-amplification mixture according to the number of reactions:

[0101]

[0102] 8) Add 45 μL of miR-Amp pre-amplification mixture and 5 μL of reverse transcription reaction product (from the previous step) to each well of the new reaction tube, with a total reaction volume of 50 μL. Slightly vortex and centrifuge. Place the reaction tube into the PCR instrument, set the reaction program according to the table below, and set the instrument to the maximum heating rate:

[0103]

[0104] 9) Prepare the qPCR reaction system. In a 1.5 mL centrifuge tube, prepare the following qPCR mixture according to the number of reactions:

[0105]

[0106] 10) Set up the following PCR reaction program:

[0107]

[0108] (7) Stability verification of candidate internal references

[0109] The Ct values ​​of the validation cohort samples detected by RT-qPCR for the candidate internal reference were imported into the online stability evaluation software RefFinder. The software will give a stability value for the copy number of each miRNA molecule in different samples. The smaller the value, the more stable the expression of the miRNA molecule.

[0110] Example 2

[0111] (1) Study cohort and clinical information

[0112] The training cohort included 103 patients, including 35 patients with benign lesions and 68 patients with malignant tumors. The validation cohort included 32 patients, including 13 patients with benign lesions and 19 patients with malignant tumors. The types of benign lesion samples included ovarian serous cystadenoma, ovarian mucinous cystadenoma, ovarian fallopian tube abscess, endometrial atypical hyperplasia, etc. The types of malignant tumor samples included low-grade and high-grade serous carcinoma and mucinous carcinoma, etc.

[0113] (2) Extraction and characterization of serum small extracellular vesicles

[0114] The present invention uses the extracellular vesicle (exosome) extraction reagent L3525 independently developed by Shanghai Siludi Biomedical Technology Co., Ltd. to extract small extracellular vesicles in the serum of ovarian cancer patients. In order to detect the characteristics of small extracellular vesicles in the serum of benign and malignant ovarian cancer patients, the present invention uses transmission electron microscopy to detect the morphology of small extracellular vesicles, and uses a fully automatic exosome fluorescence detection and analysis system to detect the expression level of small extracellular vesicle characteristic proteins. The transmission electron microscopy detection results show that the small extracellular vesicles present a typical "horseshoe-shaped" morphology, such as Figure 1 The results of the fully automatic exosome fluorescence detection and analysis system showed that the serum small extracellular vesicle characteristic proteins CD9, CD63 and CD81 were expressed in the representative samples extracted in this application, as shown in Figure 2. Figure 2 shown.

[0115] (3) Screening of candidate internal references

[0116] In the second-generation sequencing data, small extracellular vesicle miRNAs with high expression levels, small coefficient of variation and stable expression in patients with benign and malignant ovarian tumors were selected as candidate internal reference miRNAs. First, miRNAs with log2RPM values ​​<8 were eliminated. Then, the coefficient of variation (= standard deviation / mean) of each miRNA was calculated, and the coefficient of variation was sorted from small to large. The expression levels of the top 60 miRNAs were imported into the online stability evaluation software RefFinder for analysis. The analysis results are shown in Table 2. The top 6 miRNAs with the highest comprehensive stability, namely miR-30e-5p, miR-361-3p, miR-93-5p, miR-181a-5p, miR-425-5p and miR-186-5p, were selected as candidate internal references to be verified. In addition, miR-451a, which is frequently used as an internal reference, was also included in the candidate internal references to be analyzed. The detection of candidate internal references was carried out by Advanced miRNA Assay (AppliedBiosystems TM ), as shown in Table 1:

[0117] Table 1 Candidate internal reference molecule miRNA Assay information

[0118]

[0119]

[0120] (4) Stability verification of candidate internal references

[0121] In order to verify the stability of expression of the above 7 small extracellular vesicle miRNA candidate internal reference genes in ovarian tumor patients, another group of independent samples was selected as the validation cohort. RT-qPCR detection was performed in the serum small extracellular vesicles of 13 patients with benign ovarian lesions and 19 patients with malignant ovarian tumors. The expression level of each candidate internal reference was expressed as Ct value. Figure 3-Figure 9 The results showed that all candidate miRNA molecules were within the Ct value range suitable for use as internal references, among which miR-451a had the highest expression level and miR-361-3p had the lowest expression level. The expression level variation of miR-451a between different samples was the largest, and the expression level variation of the other 6 miRNAs screened by second-generation sequencing was not much different, such as Figure 3-9 The Ct values ​​of the seven candidate internal references for each sample were imported into the online stability evaluation software RefFinder. The results showed that the three analysis methods, geNorm, NormFinder and Delta Ct, all indicated that miR-30e-5p, miR-186-5p and miR-93-5p were the three most stable candidate internal reference molecules, especially the arithmetic mean values ​​of miR-30e-5p and miR-186-5p were the most stable (the smaller the value, the more stable it is, and the more stable it is, the more suitable it is to be used as an internal reference), as shown in Figure 2. Figure 10-14 As shown, this is completely consistent with the results of the second-generation sequencing data based on Genorm analysis, indicating that the reference gene combination composed of miR-30e-5p and miR-186-5p is highly stable and can be used to correct the deviations in the experimental process between different samples, thereby improving the accuracy and reliability of ovarian tumor exosome miRNA as a diagnostic biomarker.

[0122] Table 2 Stability analysis results of four algorithms

[0123]

[0124]

[0125]

[0126] Of course, it should be noted that in the above examples, the reagent extraction method is described as an example, and the extraction reagent is L3525. However, it should be appreciated that the extraction reagent can also be other commercial exosome extraction reagents, such as ExoQuick exosome precipitation solution (SBI, EXOQ5A-1), Invitrogen TM TotalExosome Isolation Kit (Life Technologies, 4484450), Exosome Serum / Plasma Kit (Qiagen, 76603). The present invention is not limited thereto. And the method for extracting serum small extracellular vesicles is not limited to the reagent extraction method, and can also be any one of ultracentrifugation extraction method, density gradient centrifugation extraction method, ultrafiltration centrifugation extraction method, and magnetic bead immunoextraction method.

[0127] In addition, in other embodiments, a detection reagent is also provided, including the PCR internal reference suitable for the serum small extracellular vesicle miRNA of ovarian tumor patients according to the above embodiment.

[0128] In addition, in other embodiments, a detection kit is also provided, comprising the aforementioned detection reagent.

[0129] In addition, in other embodiments, a method for using the aforementioned detection kit in RT-qPCR quantification of ovarian tumors is provided.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of a miRNA internal reference in the preparation of an RT-qPCR quantitative kit for serum small extracellular vesicle miRNA in patients with ovarian tumors, It is characterized in that For RT-qPCR quantification of ovarian tumors, the internal reference is a combination of miR-30e-5p and miR-186-5p; the nucleotide sequence of the miR-30e-5p is shown in SEQ ID NO.1; the nucleotide sequence of the miR-186-5p is shown in SEQ ID NO.6.

Images