siRNA, recombinant vector and application thereof for reducing rogdi gene expression
A technology of gene expression and nucleotide sequence, which is applied to siRNA, recombinant vector and its application fields to reduce the expression of ROGDI gene, and can solve the problem that the biological function of ROGDI has not been reported.
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Embodiment 1
[0044] This example is used to illustrate the high expression of ROGDI in breast cancer cell lines.
[0045] RNA extraction and reverse transcription quantitative PCR (RT-qPCR).
[0046] 1. Extraction of total RNA: this embodiment is carried out at low temperature. Cell culture was carried out in a 6-well plate, remove the medium, rinse with PBS 3 times, add Trizol 1000μl to each well, shake for 10min; collect into 1.5ml centrifuge tube, add 200μl chloroform to each tube, mix vigorously for 30sec, let stand for 15min , centrifuge at 12000rpm at 4°C for 15min; gently pipette 400μl of the upper layer into another new centrifuge tube, add an equal volume of isopropanol, gently invert and mix well, and centrifuge at 12000rpm at 4°C for 10min; discard the supernatant, and add 1ml of 75% alcohol to wash the precipitate centrifuge at 12,000 rpm at 4°C for 10 min; discard the supernatant as much as possible, and dry at room temperature for 10 min, add 10 μl of RNase-free water to eac...
Embodiment 2
[0054] This example is used to illustrate that knocking down the expression of ROGDI gene can inhibit the proliferation and / or growth of breast cancer.
[0055] 1. Preparation of RNAi lentiviral clones
[0056] 1. Target design
[0057] For the ROGDI gene sequence, according to the RNAi sequence design principles, design 3 RNAi target sequences, the nucleotide sequences of the 3 siRNAs of the ROGDI gene, and the nucleotide sequences of the negative control (NC). The names of the three siRNAs and the negative control are ROGDI-si-1a, ROGDI-si-1b, ROGDI-si-2a, ROGDI-si-2b, ROGDI-si-3a, ROGDI-si-3b, NC-si -a and NC-si-b are sequences designed for the negative control group. See Table 2 for details.
[0058] Table 2 Nucleotide sequences of 3 RNA interference targets (siRNA) and negative controls
[0059]
[0060]
[0061] Table 3 shows the nucleotide sequences of the three shRNAs used in the examples. The shRNAs used in the examples are ROGDI-sh-1a, ROGDI-sh-1b, ROGDI-sh...
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