[0036] From 2013 to 2015, the inventor collected a large number of venous serum samples from patients with esophageal squamous cell carcinoma and normal people from the First Affiliated Hospital of Nanjing Medical University and Changzhou First People's Hospital. Samples from 200 esophageal squamous cell carcinomas and 188 normal controls were used as the experimental samples for the primary screening of Exiqon miRNA qPCR panel chips and a series of subsequent qRT-PCR verifications. At the same time, 36 cases of esophageal squamous cell carcinoma tissues and 36 cases of adjacent tissues were collected. The selected patients' serum samples were all from patients with esophageal squamous cell carcinoma confirmed by pathology without surgery or intervention by radiotherapy and chemotherapy. The demographic and clinical data of these samples were systematically collected.
[0037] Refer to the flowchart ( figure 1 ), 30 esophageal squamous cell carcinoma samples and 10 normal controls were randomly selected from esophageal squamous cell carcinoma and normal control serum samples, and were mixed into 3 esophageal squamous cell carcinoma serum mixed samples and 1 normal mixed sample (one mixed sample 2ml samples were pooled from 10 200ul serum samples). The 4 mixed samples were initially screened and analyzed by Exiqon miRNA qPCR panel chip. For specific steps, refer to the instructions of Exiqon miRNA qPCR panel chip:
[0038] 1. Serum extraction
[0039] Serum samples were taken out and centrifuged at 3000 x g for 5 min after thawing to remove some debris and some insoluble components. Transfer 250ul of supernatant to a new 1.5ml tube, add 750ul of TRIZOL-LS, and shake vigorously for 5s.
[0040] 2. Two-phase separation
[0041] After homogenization, samples were incubated at 15 to 30°C for 5 minutes. Add 0.2 ml of chloroform to each 1 ml of TRIZOL-LS reagent homogenized sample, and close the tube cap tightly. After vigorously shaking the tube by hand for 15 seconds, incubate at 15 to 30°C for 2 to 3 minutes. Centrifuge at 13,000g for 15 minutes at 4°C.
[0042] 3. RNA Precipitation
[0043] Transfer the aqueous phase to a new centrifuge tube. The aqueous phase was mixed with isopropanol to precipitate the RNA in it, and the amount of isopropanol added was: 1ml of TRIZOL-LS reagent homogenized sample was added with 0.5ml of isopropanol and 5ul of glycogen. Let stand at 4°C for half an hour to allow the RNA to precipitate out as much as possible. Centrifuge at 13,000 g for 15 minutes at 4°C.
[0044] 4. RNA Washing
[0045] The supernatant was removed, and at least 1 ml of 75% (v/v) ethanol was added to each 1 ml of TRIZOL-LS reagent homogenized sample to wash the RNA pellet. Let stand for 10 minutes, then centrifuge at 10,000 g for 5 minutes at 4°C.
[0046] 5. Redissolving the RNA Precipitate
[0047] Remove the ethanol solution, dry the RNA pellet in air for 5-10 minutes, add RNase-free water and repeatedly pipet with a gun several times, then incubate at 55 to 60°C for 10 minutes.
[0048] 6. Measure the concentration:
[0049] Typically ~5μg RNA/50ml serum is obtained.
[0050] 7. cDNA Synthesis
[0051] (1) Dilution of template RNA: Use DEPC water to dilute 20-25ng of template RNA to 14ul (final concentration is 1.492-1.786ng/μl).
[0052] (2) Prepare the reaction solution: dissolve 5×Reaction Buffer and DEPC water on ice, and shake to mix. The Enzyme mix was placed in an ice box at -20°C, and then placed on ice before use. All reagents were used after centrifugation.
[0053] (3) Configure the reaction solution: configure the reaction solution in the following table
[0054]
[0055] (4) Mix and centrifuge the reagents: shake or aspirate the mixed reaction solution and then centrifuge to ensure that all solutions are thoroughly mixed.
[0056] (5) Reverse transcription reaction and heat inactivation: After incubating the reaction solution at 42° C. for 60 minutes, the reaction solution was incubated at 95° C. for 5 minutes to inactivate reverse transcriptase.
[0057] 8. Real-Time PCR
[0058] Reagents:
[0059] Nuclease free water(Exiqon)
[0060] SYBR TM Green master mix (Exiqon)
[0061] cDNA template
[0062] ROX (Invitrogen)
[0063] miRNA PCR ARRAY (Exiqon)
[0064] instrument:
[0065] ABI PRISM7900system (Applied Biosystems)
[0066] (1) Prepare Real-time PCR reagents: combine the prepared cDNA template, DEPC water and SYBR TM Dissolve the Green mastermix on ice for 15-20 minutes.
[0067] (2) Dilute cDNA template: Dilute the cDNA template obtained by RT reaction by 110 times with nuclease free water (for example, add 2180ul nuclease free water to 20μl reaction solution).
[0068] (3) Mix all reagents:
[0069] A. After briefly centrifuging the PCR plate, remove the sealing film.
[0070] B. Mix the 110-fold diluted cDNA template with 2×SYBR Green master mix in a 1:1 volume ratio.
[0071] C. Invert and mix the reaction solution and centrifuge
[0072] D. Add the mixed reaction solution to each well in the plate
[0073] E. Reseal the PCR plate
[0074] (4) Simple low-temperature centrifugation of the PCR plate
[0075] (5) Real-time PCR amplification: Real-time PCR amplification and melting curve analysis were performed according to the reaction conditions in the following table.
[0076] Real-time PCR cycling conditions are as follows:
[0077]
[0078] Data Analysis: Using the ΔΔCt method
[0079] Use the software provided with the PCR instrument to perform preliminary data analysis to obtain the original Cq value (Cp or Ct, the name may vary depending on the instrument).
[0080] We recommend using GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis) for standard and in-depth data analysis.
[0081] a. Calculate the ΔCt for each pathway-related gene in each treatment group.
[0082] ΔCt(group 1)=average Ct–average of HK genes’Ct for group 1 array
[0083] ΔCt(group 2)=average Ct–average of HK genes’Ct for group 2array
[0084] b. Calculate the ΔΔCt for each gene in 2 PCR Arrays (or two sets).
[0085] ΔΔCt=ΔCt(Group 2)−ΔCt(Group 1)
[0086] Remarks: Usually group 1 is the control group and group 2 is the experimental group.
[0087] c. Calculate the expression difference of the corresponding genes in group 2 and group 1 by 2-ΔΔCt.
[0088] After the initial screening of the chip, 36 differentially expressed miRNAs were obtained as shown in the following table (the difference in the three esophageal squamous cell carcinoma serum mixed samples was more than 1.5 times compared with the normal samples).
[0089]
[0090]
[0091] For the 36 differentially expressed miRNAs obtained from the initial screening, through the training set, the validation set and the additional validation set, the relative quantitative method based on qRT-PCR was used for verification. The specific steps are as follows:
[0092] 1. Serum RNA extraction: Select ABI company serum RNA extraction kit (AM1556), refer to the kit instructions, extract 200ul of each sample to extract RNA, and finally dissolve with 100ul of DEPC water.
[0093] 2. Preparation of cDNA:
[0094] 1) Use a 50 μL reaction system for reverse transcription experiments
[0095]
[0096] The above reaction system was mixed and centrifuged briefly, and the reaction was carried out according to the following procedure:
[0097]
[0098] 2) After the above-mentioned reaction, the following reactants are added in the reaction system again
[0099]
[0100] 3. qPCR
[0101] 1) Use a 5 μL reaction system to test in the following proportions
[0102]
[0103] The reaction system was mixed, centrifuged briefly, placed in a real-time quantitative PCR instrument, and the reaction was carried out according to the following procedures:
[0104]
[0105] Add a dissolution curve after the reaction is complete.
[0106] Data analysis: The relative concentration of miRNA in each sample can be calculated by the ΔΔCt method. SPSS 16.0 software was used for statistical analysis, and a group of 5 miRNAs that were highly expressed in the serum of esophageal squamous cell carcinoma in both the training set and the validation set were obtained: miR-20b-5p, miR-28-3p, miR-192-5p , miR-223-3p and miR-296-5p (with P values less than 0.05 in both training and validation sets, figure 2 ). From these 5 miRNAs, the ROC curve of each sample can be calculated. like image 3 , the molecular markers composed of these five miRNAs can well distinguish patients with esophageal squamous cell carcinoma from normal people.
[0107] The research group further detected the expression of these five miRNAs in esophageal squamous cell carcinoma tissues and exosomes in serum. The RNA was extracted from esophageal squamous cell carcinoma tissues using TRIZOL, and the exosome extraction kit was ExoQuick kit (SBI company). The exosomes extracted from 200ul serum were resuspended in 200ul DEPC water, and then the exosomal RNA was extracted by AM1556 kit (ABI company), and the steps were the same as the serum RNA extraction process.
[0108] Using nonparametric test analysis, it was found that the expressions of miR-20b-5p, miR-28-3p and miR-192-5p in esophageal squamous cell carcinoma tissues were higher than those in adjacent tissues ( Figure 4 ). The expression of miR-296-5p in esophageal squamous cell carcinoma serum exosomes was also significantly higher than that in the normal population ( Figure 5 ).
[0109] The kit includes a batch of serum miRNA qRT-PCR primers, as well as common reagents required for the corresponding PCR technology, such as: reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probes, RNase inhibitors, Taq Enzymes, etc., can be selected according to the specific experimental method. These commonly used reagents are well known to those skilled in the art, and there may also be standards and controls (such as quantitatively standardized normal human samples, etc.). The value of this kit is that only serum and no other tissue samples are needed, and the expression of miRNA in serum samples is detected by the most simplified fluorescence method to assist in the diagnosis of the possibility of esophageal squamous cell carcinoma in the patient from which the sample is derived. Serum miRNA detection is convenient and accurate in quantification, which greatly improves the sensitivity and specificity of disease diagnosis. Therefore, putting this kit into practice can help guide diagnosis and further individualized treatment.
