Application of human EDDM3A gene and related products
A technology of EDDM3A and gene, which is applied in the field of application of human EDDM3A gene and related products, and can solve the problem that EDDM3A reports cannot be retrieved
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Embodiment 1
[0114] Example 1 Preparation of RNAi lentivirus against human EDDM3A gene
[0115] 1. Screening for effective siRNA targets against the human EDDM3A gene
[0116] Retrieve EDDM3A (NM_006683) gene information from Genbank; design effective siRNA targets for EDDM3A gene. Table 1-1 lists the screened effective siRNA target sequences against the EDDM3A gene.
[0117] Table 1-1 is targeted at the siRNA target sequence of human EDDM3A gene
[0118] SEQ ID NO TargetSeq(5'-3') 1 GGCTGTGTGTATACAGTAA
[0119] 2. Preparation of lentiviral vector
[0120] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.
[0121] Table 1-2 Double-stranded DNA Oligo with sticky...
Embodiment 2
[0139] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency
[0140] Human gastric adenocarcinoma cells AGS and human gastric cancer cells MGC80-3 in the logarithmic growth phase were respectively digested with trypsin to make cell suspensions (the number of cells was about 5×10 4 / ml) were inoculated in 6-well plates, and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI: 10 for AGS, 20 for MGC80-3), add an appropriate amount of the lentivirus prepared in Example 1, culture for 24 hours, replace the medium, and collect the cells after the infection time reaches 5 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for...
Embodiment 3
[0147] Example 3 Western Blotting method to detect gene silencing efficiency
[0148] 1. Extraction of total cell protein
[0149] 1) Infect the target cells (AGS and MGC80-3 cells) with the control virus and the RNAi virus targeting the EDDM3A interference target according to the multiplicity of infection (MOI: 10 for AGS, 20 for MGC80-3).
[0150] 2) After 5 days of infection, the cell samples were collected and washed twice with PBS. Take an appropriate amount of RIPA lysate, add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM. (Use RIPA lysate, instruction link: http: / / www.beyotime.com / ripa-lysis-bufferm.htm).
[0151] 3) Add an appropriate amount of RIPA lysate, and lyse on ice for 10-15 minutes. Scrape the cells and transfer them into a new EP tube, and then ultrasonically disrupt the cells (20 times at 40W, 1 s each time, 2 s apart).
[0152] 4) Centrifuge at 12000g for 15min at 4°C, take the supernatant and use BCA Protein Assay...
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