The inhibitor DDX3 used in our laboratory was Human shDDX3 of lentivirus, and the control group was shLuc of lentivirus, which was transfected into the cells by infection.
First, BHK-21 cell was cultured to 6 cm dish and cultured to a cell density of about 12%, then remove the medium and rinse twice with 1 ml of PBS.
After removing the PBS, then add lentivirus (shDDX3 and shLuc) (MOI = 50) and 3ml 2% RPMI (containing 1/1000 polybrene), then culture for 24 hours at 37 ℃ with 5% carbon dioxide (CO2) incubator.
The next day, adding 10μM / ml puromycin effect for 24 hours, then infect JEV T1P1 strain for 24hr and 36hr. Final, collecting the cell (24 hours) and received supernatant (24 hours and 36 hours).
Then a host protein and JEV protein were …show more content…
First, 0.5 ml of TRlzol reagent was added to the culture dish and the cells were scraped from the culture dish with a cell spatula, taken up to a new Eppendorf, stand at room temperature for 5 minutes, then add 0.1 ml of chloroform and about 15 seconds with gentle agitation (pink color).Stay at room temperature for 3 minutes and centrifuge 12,000 rpm, 4 ℃, 15 …show more content…
First, adding 2ul of 5 times gDNA eraser, 3ul of DEPC water and 5 μl of RNA solution into PCR tube, then heated at 42 ° C for 3 minutes in an ABI PCR machine and then placed on ice for a while.
Adding 2 μl of RT buffer, 1 μl of RT enzyme mix, 2 μl of RT primer and 5 μl of DEPC water into PCR tube, then heated to 42 ° C, 15 minutes, 95 ° C for 3 minutes and placed on ice for a 1 hr. cDNA was divided into three groups, one group was positive viral RNA, negative viral RNA, and internal control. Adding 2 μl of cDNA, 0.4 μl of Forward primer（viral RNA：JEV-3642- Forward primer； internal control：miGAPDH- Forward primer）, 0.4 μl of reverse primer（viral RNA： JEV-3801-Reverse primer； internal control： miGAPDH- Reverse primer, 0.4 μl of Rox low dye, 10 μl of 2 times SYBR green fast qPCR mix and 6.8 μl of DEPC water.
According to the procedure of Real-time qPCR, the reaction was carried out at 95 ° C for 20 seconds, 95 ° C for 3 seconds, 60 ° C for 30 seconds, 95 ° C for 15 seconds, and 60 ° C for 1 minute for 40 cycles.
We Use the 7500 Software v2.0.4 (Applied Biosystems ™, ABI) to analyze the
First, BHK-21 cell was cultured to 6 cm dish and cultured to a cell density of about 12%, then remove the medium and rinse twice with 1 ml of PBS.
After removing the PBS, then add lentivirus (shDDX3 and shLuc) (MOI = 50) and 3ml 2% RPMI (containing 1/1000 polybrene), then culture for 24 hours at 37 ℃ with 5% carbon dioxide (CO2) incubator.
The next day, adding 10μM / ml puromycin effect for 24 hours, then infect JEV T1P1 strain for 24hr and 36hr. Final, collecting the cell (24 hours) and received supernatant (24 hours and 36 hours).
Then a host protein and JEV protein were …show more content…
First, 0.5 ml of TRlzol reagent was added to the culture dish and the cells were scraped from the culture dish with a cell spatula, taken up to a new Eppendorf, stand at room temperature for 5 minutes, then add 0.1 ml of chloroform and about 15 seconds with gentle agitation (pink color).Stay at room temperature for 3 minutes and centrifuge 12,000 rpm, 4 ℃, 15 …show more content…
First, adding 2ul of 5 times gDNA eraser, 3ul of DEPC water and 5 μl of RNA solution into PCR tube, then heated at 42 ° C for 3 minutes in an ABI PCR machine and then placed on ice for a while.
Adding 2 μl of RT buffer, 1 μl of RT enzyme mix, 2 μl of RT primer and 5 μl of DEPC water into PCR tube, then heated to 42 ° C, 15 minutes, 95 ° C for 3 minutes and placed on ice for a 1 hr. cDNA was divided into three groups, one group was positive viral RNA, negative viral RNA, and internal control. Adding 2 μl of cDNA, 0.4 μl of Forward primer（viral RNA：JEV-3642- Forward primer； internal control：miGAPDH- Forward primer）, 0.4 μl of reverse primer（viral RNA： JEV-3801-Reverse primer； internal control： miGAPDH- Reverse primer, 0.4 μl of Rox low dye, 10 μl of 2 times SYBR green fast qPCR mix and 6.8 μl of DEPC water.
According to the procedure of Real-time qPCR, the reaction was carried out at 95 ° C for 20 seconds, 95 ° C for 3 seconds, 60 ° C for 30 seconds, 95 ° C for 15 seconds, and 60 ° C for 1 minute for 40 cycles.
We Use the 7500 Software v2.0.4 (Applied Biosystems ™, ABI) to analyze the