Computational protocol: Pathogenesis of Autosomal Dominant Hereditary Spastic Paraplegia (SPG6) Revealed by a Rat Model

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Protocol publication

[…] Poly(A)-RNA was harvested from organs of 3-week-old Tg and wild-type rats using STAT-60 (Tel-Test, Friendswood, TX) and Qiagen RNA extraction kit (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) primer sequences and the detailed protocol for RT-PCR are described in Supplemental Digital Content 1, http://links.lww.com/NEN/A510. ABI-prism 7700 HT (Applied Biosystems, Foster City, CA) was used for quantitative PCR analysis. For protein detection, protein extracts were applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis using anti-NIPA1, anti-BMPRII, anti–phosphorylated Smad 1/5/8, and anti–β-actin antibodies. ImageJ Software was used for quantification of proteins. [...] Three Tg and 3 age-matched wild-type rats, at 38 weeks of age, were used for quantitative analysis. Five randomly selected areas of 1-μm-thick toluidine blue–stained cross sections from midsciatic and distal tibial nerves were photographed using a 60× objective. Axon diameters and numbers of myelinated nerves were determined using Adobe Illustrator CS4 software (Adobe Systems Inc., San Jose, CA). The myelinated fiber densities (mean ± SD per square millimeter of endoneurial area) in each group were calculated, and axon diameter histograms were generated. A total of 2,080 and 6,180 fibers were counted for each genotype (wild-type and Tg, respectively). […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus, Homo sapiens
Diseases Peripheral Nervous System Diseases, Prader-Willi Syndrome, Spastic Paraplegia, Hereditary