Computational protocol: Regulation of Shootin1 Gene Expression Involves NGF-induced Alternative Splicing during Neuronal Differentiation of PC12 Cells

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[…] Growing cells were plated on collagen type IV from human placenta pre-coated 60 mm dishes. Adhered cells then left untransfected or transfected with NT-shRNA, shRNA-I and shRNA-II, separately. Cells were then analyzed by phase contrast microscopy and counted for neurite outgrowth. For the length measurements, the neurites equal to or greater than a cell body in length were considered. The lengths were measured as the distance between the edge of a cell body and the tip of a growth cone. Only clearly visible cells were subjected to analysis to prevent inaccurate scoring. Images of 100–120 neurons per sample group were captured for each experiment. Neurite lengths were measured by using NeuronJ (ImageJ plug-in; NIH, Bethesda, MD, USA). The average neurite length was obtained by dividing the total neurite length by the number of cells and expressed as % control to yield the average neurite length per sample group. [...] The promoter and cDNA sequences of shootin1 (RGD1311558; Gene ID: 292139) retrieved from Genbank (Rnor_5.0 r.104). Primers were designed with Primer3 software to detect the constitutive and alternative splicing products. To facilitate methylation detection, bisulfite PCR primers were designed by using the Methyl Primer Express Software v1.0 (Applied Biosystems). Comparative analysis of shootin1 protein variants for the regions that are recognized by shootin1 antibody in rat, were done using the BLASTp program. […]

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