MM (multiple myeloma)-resistant small-interference RNA (ribonucleic acid) and application thereof
A multiple myeloma, ribonucleic acid technology, applied in the fields of application, antineoplastic drugs, medical preparations containing active ingredients, etc., can solve the problem of lack of effective therapeutic molecular targets
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Embodiment 1
[0052] Example 1: Design and synthesis of siRNA against SUMO-1 gene
[0053] Design and synthesize SUMO-1 small interfering RNA (siRNA), wherein the target sequence in the SUMO-1 gene of the siRNA is as shown in Table 1:
[0054]siRNA target sequence (CDNA) of table 1 SUMO-1 gene
[0055] serial number
serial number
sequence
siRNA-1
SEQ ID NO:1
CTGGGAATGGAGGAAGAAG
siRNA-2
SEQ ID NO:2
CAATGAATTCACTCAGGTT
siRNA-3
SEQ ID NO:3
GGACAGGATAGCAGTGAGA
[0056] According to the sequence in the above table, the siRNA sequence was synthesized by Ribo (Guangzhou) Company, and ddH 2 O dilute the synthesized siRNA nucleotide molecules to a concentration of 100 μM for later use. The sequence information and structural information of the resulting siRNA are as follows:
[0057] Fragment sequence of siRNA-1: sense strand 5'CUGGGAAUGGAGGAAGAAG dTdT 3' (as shown in SEQ ID NO:4 in the sequence listing);
[0058] Antisense st...
Embodiment 2
[0063] Example 2: Using siRNA to transfect myeloma cells
[0064] Myeloma cells NCI-H929 (the cells were purchased from ADCC) and RPMI-8226 (the cells were purchased from ADCC) in the logarithmic growth phase were centrifuged for 5 min, the supernatant was discarded, and the cells were resuspended with PBS, and then Centrifuge for 5min. Use the ELX800 microplate reader to adjust the cell concentration to 30×10 4 cells / L, inoculated into a six-well plate overnight. The cationic liposome was lipo2000, and the siRNA-3 obtained in Example 1 was dissolved in water, and the final concentration of the obtained aqueous solution was adjusted to 100 nM. The cationic liposome and siRNA solutions were diluted with optimum respectively, and left at room temperature for 5 minutes for later use. Mix the cationic liposome solution and the siRNA solution so that the liposome fully encapsulates the siRNA, and let it stand at room temperature for later use. After 20 min, add the mixture to t...
Embodiment 3
[0083] Example 3: Effect of SUMO-1 Gene Expression Inhibition on Apoptotic Rates of Different Multiple Myeloma Cells
[0084] Annexin V-FITC-PI kit (purchased from KGI, Shanghai) and flow cytometry (model BD FACS calibur 4, USA) were used for detection.
[0085] Flow cytometry: Suspended cells were collected by centrifugation (2000rpm for 5min); cells were washed twice with PBS (2000rpm for 5min) to collect 1-5×10 5 Cells; add 500 μL Binding Buffer to suspend cells; add 5 μL Annexin V-FITC and mix well, then add 5 μL Propidium Iodide, mix well; room temperature, dark, react for 5-15 minutes; flow cytometry detection.
[0086] MTS method: divide cells into 10 × 10 3Inoculate each well in a 96-well plate, add 20 μl MTS (purchased from Promega) after 48 hours of treatment, 37 ° C, 5% CO 2 After incubating in the cell culture box for 2 hours, use a Flex Station 3 (purchased from Molecular Devices) microplate reader to detect (490 / 690nm).
[0087] From the target polynucleotide ...
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