Circular RNA marker for detecting Crohn's disease and application
A Crohn's disease and marker technology, applied in the field of molecular biomedicine, can solve the problems of low sensitivity and specificity
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Embodiment 1
[0030] Example 1 CD Peripheral Blood Mononuclear Cell Separation
[0031] Peripheral blood was collected from patients with Crohn's disease and healthy controls using EDTA-K2 anticoagulant tubes, and 6 mL of blood was collected from each case. At the time of sample collection, all participants had not used immunosuppressants and oral antibiotics within 3 months, and had no other chronic diseases. All patients with Crohn's disease were diagnosed by endoscopy, and ulcerative colitis, Behcet's disease, and irritable bowel syndrome were excluded. The healthy control subjects had no gastrointestinal diseases and other chronic diseases in the physical examination center of the hospital, and all indicators were normal before sampling. After the sample is collected, it is quickly sent to the central laboratory for the isolation of peripheral blood mononuclear cells, and the isolation is completed within 2 hours. Peripheral blood mononuclear cells were separated by the Ficoll method,...
Embodiment 2
[0044] Example 2 Total RNA extraction
[0045] 2.1 Collect cells by centrifugation, add 1 ml of TRIzol to every 5-10×106 cells, and pipette repeatedly. Do not wash cells before adding TRIzol to avoid mRNA degradation.
[0046] 2.2 Put the sample at room temperature (15-30°C) for 5 minutes to completely separate the nucleic acid-protein complexes.
[0047] 2.3 Add 0.2 ml of chloroform for every 1 ml of TRIzol used, shake vigorously for 15 seconds, and place at room temperature for 3 minutes.
[0048] 2.4 Centrifuge at 10000×g at 2-8°C for 15 minutes. The sample is divided into three layers: the bottom layer is the yellow organic phase, the upper layer is the colorless aqueous phase and an intermediate layer. The RNA is mainly in the aqueous phase, which is about 60% of the volume of the TRIzol reagent used.
[0049] 2.5 Transfer the aqueous phase to a new tube. If you want to separate DNA and protein, you can keep the organic phase. See below for further operations. RNA in...
Embodiment 3
[0053] Example 3 Reverse transcription and quantitative detection of hsa_circRNA_009084 qPCR
[0054] with PrimeScript TM The Master Mix (TaKaRa, Shiga, Japan) kit reverse transcribed RNA into cDNA. With β-actin as internal reference, TB Green TM Premix Ex Taq TM II (TliRNaseH Plus; TaKaRa, Shiga, Japan) kit dye method qPCR was used to detect the relative expression levels of target circRNA molecules in Crohn's disease patient group and control group samples.
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