br Materials and methods br Cell culture and reagents br
2. Materials and methods
2.1. Cell culture and reagents
Cells were seeded into 96-well plates in triplicates (2000 cells/well). The number of Necrostatin 1 were measured by Celigo Imaging Cytometer for three to five days. The numbers were normalized to the cell number on day-1 (the next day after seeding) and plotted over time.
For MTT assay, 20 μl 5 mg/ml MTT were added to the cells for the last four hours. Then culture media was removed and 150 μl DMSO was added into each well. The plate was shaken for 2–5 min, and sample absorbance was measured at 490 nm/570 nm using Tecan Infinite M2009PR plate reader.
2.3. Knocking down of SNRPA1 by lentiviral mediated shRNA
2.3.1. SiRNA sequences selecting and shRNA oligo designing
Choosing siRNA sequences with target SNRPA1 is critical. Based on the online siRNA designing tools (http://www.ambion.com/techlib/ misc/siRNA_finder.html), a couple of candidate siRNA sequences were prescreened and their gene silencing eﬃciency were validated. Finally, siRNA with sequence as TGGTATAATAGCCTTGTTT were selected in this study. The corresponding shRNA oligonucleotides were then syn-thesized and purified by GENERAY Co., Ltd, Shanghai. Followed by annealing the shRNA oligos, they were inserted between AgeI and EcoRI restriction enzyme sites into the lentiviral vector GV115. Ligated lentiviral vector containing target shRNA inserts were then transformed into TOP10 competent cells (TIANGEN, China) and recombinant clones were identified by standard PCR amplification and restriction enzyme digestion as well as DNA sequencing. A large-scale plasmid prep from the identified positive clone were made according to the EndoFree Maxi plasmid kit manufacturer’s protocol (TIANGEN, China).
2.3.2. Production of shRNA lentivirus using packaging plasmids Approximately 24 h before transfection, 5 × 106 HEK293T cells
were seeded into 10 cm tissue culture plates in 15 ml growth medium (DMEM + 10% FBS) and incubated at 37 °C, 5% CO2 overnight. 2 h before transfection, complete growth medium was replaced with DMEM only without FBS. Transfection of DNA mixture of packing plasmids and lentivirus vectors was performed when cells were about 70%–80% confluent. About 6 h after transfection, the transfection medium was replaced with 20 ml fresh complete growth medium and cells were incubated at 37 °C for additional 48–72 h. At about 48 h after the start of transfection, Lentiviral supernatants were harvested, centrifuged at 4000g for 10 min and supernatants were filtered through a 0.45 μm low protein binding filter to remove cellular debris. shRNA lentiviruses were concentrated by ultracentrifugation (25,000 rpm × 2 h, 4 °C) and the virus pellets were resuspended in PBS/0.1% BSA. After virus is fully dissolved, it’s centrifuged at 10,000 rpm for 5 min and aliquoted for future use. Viral titers were set to 3 × 108 transduction units (TU)/ml.
complete growth medium (RPMI1640 + 10% FBS) and incubate the cells at 37 °C, 5% CO2 for 12–24 h before infection. For both the control and shRNA lentivirus gene silencing groups, A total of 100 μl of lenti-viral particles (PSC14835, LVpFU-GW-012PSC66103-1, respectively) with a virus titer of 3 × 108/ml prepared from the step above were used to infect 5 culture plates each containing 1.2 × 106 cells to give a MOI of 5 for less than 8 h. Remove and discard the virus-containing trans-duction medium and add fresh growth medium after 12 h past the in-itial infection time point. Continue to incubate the cells for additional 24–48 h to allow the shRNA to reach its maximum eﬀect. Fluorescent images of GFP were taken at 72 h after initial infection to assess the lentivirus transducing eﬃciency. Puromycin were added at 4 μg/ml at 72 h post infection for additional 48 h. Culture of Infected cells were continued until 120 h post infection. At 144 h post infection, cells were seeded and cultured with proper concentrations for later experiments.
2.4. SNRPA1 gene expression analysis by qRT-PCR and Western Blotting (WB)
To technically validate the gene silencing eﬃciency of SNRPA1 by shRNA lentivirus, gene transcription of SNRPA1 was analyzed by qRT-PCR. cDNA was synthesized from 1ug total RNA samples using Promega M-MLV cDNA kit. qPCR was performed in Agilent MX3000p Real-time PCR. Universal reaction and amplification conditions were used. GAPDH was the endogenous control and Ct method was used to quantify the relative amount of mRNA in each sample in comparison with the control. Additionally, qPCR was conducted to assess the gene expression of selected candidate genes regulated by SNRPA1. Similar procedures were used. Each experiment was done in either duplicate or triplicate, and the average was calculated. Primer sequences used are: