Computational protocol: Discovery of a Dicer-Independent, Cell-Type Dependent Alternate Targeting Sequence Generator: Implications in Gene Silencing & Pooled RNAi Screens

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[…] Cells were lysed with RIPA buffer (Sigma-Aldrich) containing 1 mM phenylmethylsulfonyl fluoride and 2 mM tris(2-carboxyethyl)phosphine hydrochloride by sonication at 60 amplitude for 10 min on a water/ice mix. Cellular debris was removed by centrifugation at 13,000 rpm for 30 min. 20 µg for HCT116, and 40 µg for RKO and DLD1 samples were separated in 3–8% tris-acetate gels at 150 V for 55 min (BIORAD, Berkeley, CA) and transferred onto PVDF membrane (Millipore, Billerica, MA) at 4°C and 100 V for 2 h. Membranes were blocked with 5% non fat dry milk (BIORAD) in PBS containing 0.1% Tween-20 for 1 h at room temperature. The membranes were incubated independently with anti-DICER1 antibody (1∶500, Abcam, Cambridge, MA) and anti-Actin (1∶400, Abcam) overnight at 4°C, followed by 1 h incubation at room temperature with horseradish peroxidase-conjugated secondary antibody (1∶5000, Cell Signaling, Danvers, MA). The images were analyzed and quantified using ImageJ. [...] For the purpose of evaluating the activity of individual siRNA duplexes in the experiment, the data values corresponding to the gain in EGFP fluorescence intensity were obtained and converted into percentage gain (% gain) relative to the Silencer Select Negative Control No. 1 calculated using the following formula: The threshold was determined at a % gain in EGFP signal to be at least 2 standard deviations above the mean of the negative control. In parallel, the cytotoxicity of individual siRNA duplexes was determined using the residual nuclei count (NUCL), also converted into percentage inhibition (% inhib) calculated using the following formula:Where, the high control is Silencer Select Negative Control No. 1 and the low control is PLK1 (s449). The threshold to score for cytotoxic siRNA duplexes was set at atleast 50% kill relative to the controls. All siRNA duplexes were screened in quadruplicates and the corresponding values were averaged for calculating a single % gain and % inhib for each siRNA. The data analysis was done using Perl scripts and the graphical representation for data visualization was done using SigmaPlot (Systat Software Inc., San Jose, CA). […]

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