MiRNA-21 detection method
A miRNA-21 and detection method technology, which is applied in the field of microRNA detection, can solve the problems of complex operation, long detection time, and high requirements for operation skills, and achieves the effects of strong modifiability, low cost, and improved detection sensitivity.
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Embodiment 1
[0038] Example 1: Successful verification of substrate chain-sucrase complex and probe synthesis
[0039] 1. Preparation of substrate chain and sucrase linker complex:
[0040] Prepare 30 μL of substrate chain with a concentration of 1 mM in DEPC water, add 2 μL of sodium phosphate buffer (1M) at pH 5.5 and 2 μL of tris(2-carboxyethyl)phosphine (TCEP) at a concentration of 30 mM dissolved in Millipore water, and The mixture was allowed to stand at room temperature for 1 hour and then purified by ultrafiltration 8 times using Amicon-10K ultrafiltration tubes.
[0041] Sucrase-linked substrate chains: Prepare 400 μL of 20 mg / mL sucrase solution with buffer A, mix with 1 mg sulfo-SMCC; vortex for 5 minutes, place on a shaker at room temperature for 1 hour, and centrifuge at 1000r for 5 minutes Excess sulfo-SMCC was removed and the solution was purified by ultrafiltration 8 times with Amicon-100K and buffer A.
[0042] The purified sulfo-SMCC-activated sucrase solution was mixed...
Embodiment 2
[0051] Example 2: Detection sensitivity experiment of miRNA-21
[0052] 1. The instruments and reagents required for the experiment are the same as those in Implementation 1;
[0053] 2. Investigate the sensitivity of the deoxyribozyme and sucrase dual-enzyme amplification system to measure the miRNA-21 standard solution, including the following steps:
[0054] (1) Preparation of samples: prepare miRNA-21 standard solutions of 10pM-200pM concentration with DEPC water, so that the concentrations are 10pM, 20pM, 50pM, 100pM, 150pM, 200pM;
[0055] (2) Perform the reaction: put 20uL detection probe in the magnetic rack for 2 minutes, discard the clear solution, then add 10μL different concentrations of miRNA-21 to be detected (SEQ ID NO.2), 7μL DEPC-treated water, 2μL double Strand-specific nuclease buffer (containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2 and 1 mM DTT) and 1 μL DSN (1 U / uL). And react at 45°C for 50 minutes. The miRNA-21 to be tested specifically binds to the hyb...
Embodiment 3
[0061] Embodiment 3: Investigate the selectivity to miRNA-21
[0062] 1. The instruments, reagents and probe synthesis methods required for the experiment are consistent with those in Implementation 1;
[0063] 2. The sequences required for this experiment, in addition to the sequences applying for rights protection, also need to use the following sequences (as shown in Table 1):
[0064] Table 1 List of oligonucleotide sequences
[0065]
[0066]
[0067] 3. Deoxyribozyme and sucrase-based dual-enzyme amplification system measures the selectivity of miRNA-21 and mismatched sequences, including the following steps:
[0068] (1) Prepare samples: Prepare hsa-miR-200c, hsa-mir-423, hsa-miR-4640, has-miR-21, Mis-1, Mis-2, Mis-3 and NC sequences with DEPC water 200pM standard solution, blank control (Blank) is DEPC water;
[0069] (2) Perform the reaction: put 20 uL detection probe in the magnetic rack for 2 minutes, discard the clear solution, and then add 10 μL of differ...
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