Sicon1

Manufactured by Horizon Discovery

SiCON1 is a laboratory equipment designed for the analysis and processing of biological samples. It functions as a versatile instrument capable of performing various analytical techniques.

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Lab products found in correlation

Sicon2, by Horizon Discovery (3 mentions) Rnaimax, by Thermo Fisher Scientific (3 mentions) Incucyte s3, by Sartorius (1 mentions) Sigenome smartpool, by Horizon Discovery (1 mentions) Lipofectamine rnaimax transfection reagent, by Promega (1 mentions) Celltiter glo assay, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Sigenome smartpool sirna, by Horizon Discovery (1 mentions) Smartpool, by Horizon Discovery (1 mentions) Celltiter glo luminescence reagent, by Promega (1 mentions)

4 protocols using sicon1

High-throughput siRNA Screening in CDK12 Mutant Cells

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CDK12^as cells (1000 cells/well) were reverse transfected in a 96 well plate format with a custom siGENOME SMARTPool (Dharmacon) siRNA library as described previously^{72 (link)} using Lipofectamine RNAimax Transfection Reagent (Promega). Positive (siPLK1) and negative controls (siCON1, Dharmacon) were also included in each plate. After 24 hours, media was replaced with new media drug containing 1NM (0.3 μM) or the drug vehicle (DMSO), and cells were continuously cultured for six days further, at which point cell viability was estimated by the addition of CellTiter-glo reagent to the media for 10 minutes. Drug Effect Z scores were calculated from the resultant luminescence data as described previously^{73 (link)}. Each screen was carried out in triplicate, with the data being combined in the final analysis. For single gene siRNA experiments, C4-2B cells were plated in 96-well plates and allowed to adhere overnight. The next day, cells were transfected using siGENOME SMARTPool (Dharmacon) against the indicated genes (CCNK, CDK13) or nontargeting control (NTC) as above. The plate was then placed in an IncuCyte S3 (Sartorius) and cell growth monitored over the indicated time frame.

Tien J.C., Chang Y., Zhang Y., Chou J., Cheng Y., Wang X., Yang J., Mannan R., Shah P., Wang X.M., Todd A.J., Eyunni S., Cheng C., Rebernick R.J., Xiao L., Bao Y., Neiswender J., Brough R., Pettitt S.J., Cao X., Miner S.J., Zhou L., Wu Y.M., Labanca E., Wang Y., Parolia A., Cieslik M., Robinson D.R., Wang Z., Feng F.Y., Lord C.J., Ding K, & Chinnaiyan A.M. (2024). CDK12 Loss Promotes Prostate Cancer Development While Exposing Vulnerabilities to Paralog-Based Synthetic Lethality. bioRxiv.

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High-throughput RNAi screening for cancer vulnerabilities

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Cells lines were reversed transfected with a Dharmacon SMARTpool 384 well plate-arrayed siRNA library designed to target 714 kinases and kinase-related genes, 320 Wnt-pathway associated genes, 80 tumour suppressor genes, and 480 genes recurrently altered in human cancers (Supplementary Table 1) as described in (20 (link)). Positive control (siPLK1) and multiple negative controls (siCON1 and siCON2; Dharmacon, catalogue numbers D-001210-01-20 and D-001206-14-20, and AllStar; QIAGEN, catalogue number 1027281) were included on every plate. Transfection reagents were as follows: SYO-1; Aska-SS; Yamato-SS and CME-1 = RNAiMAX (Invitrogen); HS-SY-II = Lipofectamine 2000 (Invitrogen). Screens were performed in triplicate. Cell viability was estimated five days after transfection using CellTiter-Glo assay (Promega). Data processing and quality controls were performed using the cellHTS2 R package as described previously (20 (link), 21 (link)). In the case of the VX970 resistance screen (Figure 4), 24h after transfection, VX970 (final concentration 0.75 μM) or vehicle (DMSO) was added to cells. Cells were then exposed to VX970 for four days and viability estimated with CellTiter-Glo assay as above. Drug effect (DE) Z-scores were calculated as described previously (22 (link)).

Jones S.E., Fleuren E.D., Frankum J., Konde A., Williamson C.T., Krastev D.B., Pemberton H.N., Campbell J., Gulati A., Elliott R., Menon M., Selfe J.L., Brough R., Pettitt S.J., Niedzwiedz W., van der Graaf W.T., Shipley J., Ashworth A, & Lord C.J. (2017). ATR is a therapeutic target in synovial sarcoma. Cancer research, 77(24), 7014-7026.

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Optimized Cell Line Maintenance and Transfection

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All cell lines were obtained from the American Type Culture Collection and maintained according to the supplier’s instructions. Cell lines were routinely STR typed and mycoplasma tested. Cell lines were grown and transfected with individual and SMARTpool siGENOME siRNA (Dharmacon) and transfected using RNAiMAX (Invitrogen) reagent as described in ref. [21 (link)]. Transfection efficiency was monitored using positive control (siPLK1) and two different negative control siRNAs (siCON1 and siCON2; Dharmacon, catalogue numbers D-001210-01-20 and D-001206-14-20). SKPinC1 inhibitor was purchased from Tocris (no. 4817) and was solubilised as a 10 mM stock solution in dimethyl sulfoxide (DMSO).

Brough R., Gulati A., Haider S., Kumar R., Campbell J., Knudsen E., Pettitt S.J., Ryan C.J, & Lord C.J. (2018). Identification of highly penetrant Rb-related synthetic lethal interactions in triple negative breast cancer. Oncogene, 37(43), 5701-5718.

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Reverse Transfection Protocol for siRNA Screening

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Reverse transfections using the siRNA SMARTpool, siCON1 and siCON2 negative controls (Dharmacon) were carried out in 384-well plates, 6-well plates or 10 cm dishes using 20 nmol/L of siRNA (unless specified), mixed with 12.5% of the final volume of optiMEM and incubated at room temperature for 10 minutes. In parallel, 20 nmol/L RNAiMax (Thermo Fisher Scientific) was added to 12.5% of the final volume of optiMEM and incubated at room temperature for 10 minutes. siRNA and RNAiMax mixtures were mixed and incubated at room temperature for 30 minutes before applying to the cells. Lysates were retrieved or viability experiments were performed after 2 to 3 days.
All viability assays were performed in triplicate using CellTiter-Glo luminescence reagent (Promega). The surviving fraction was calculated as follows: Surviving fraction = (luminescence in siRNA treated well)/(luminescence in siControl treated wells). The normalized percentage of inhibition, to normalize data between different cell lines by the efficiency of transfection, was calculated using the following formula: ((mean (positive control) − Sample)/(mean (positive control) − mean (negative control)) × 100. Supplementary Table S1 contains all the siRNAs used in this study.

Llorca-Cardenosa M.J., Aronson L.I., Krastev D.B., Nieminuszczy J., Alexander J., Song F., Dylewska M., Broderick R., Brough R., Zimmermann A., Zenke F.T., Gurel B., Riisnaes R., Ferreira A., Roumeliotis T., Choudhary J., Pettitt S.J., de Bono J., Cervantes A., Haider S., Niedzwiedz W., Lord C.J, & Chong I.Y. (2022). SMG8/SMG9 Heterodimer Loss Modulates SMG1 Kinase to Drive ATR Inhibitor Resistance. Cancer Research, 82(21), 3962-3973.

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