Computational protocol: DICER1 hotspot mutations in non-epithelial gonadal tumours

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Protocol publication

[…] DNA was extracted from FFPE tumour samples using 3–5 slides of 10 μm thick tumour tissue with the QIAamp DNA FFPE Tissue Kit (QIAGEN, Toronto, ON, Canada) according to protocol. DNA from fresh-frozen tumour tissues was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). Sanger sequencing was used to screen the RNase IIIa and IIIb domains of DICER1 in the tumour samples. Primer pairs for PCR amplification and sequencing were designed using Primer3 (http://frodo.wi.mit.edu/) to flank exons (). The sequences were then filtered using OligoCalc software () to avoid hairpin formation and UCSC in silico PCR software to ensure yield of a single product. DNA from any FFPE sample in which a mutation was found was extracted twice independently and the PCR was repeated at least twice from each independent extraction using QIAGEN HotStarTaq, 10 mM dNTP and 10 × PCR buffer reagents with 1.4 μl of 20 μM primers in a 50-μl reaction. Thermocycler parameters can be found in the . PCR products were purified and sequenced by conventional Sanger methods by the McGill University and Genome Quebec Innovation Centre. Sequences were analysed visually using Lasergene Version 10 (DNASTAR, Madison, WI, USA). […]

Pipeline specifications

Software tools Primer3, OligoCalc
Application qPCR
Organisms Homo sapiens
Diseases Neoplasms