Computational protocol: Mutation of TGFβ-RII eliminates NSAID cancer chemoprevention

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Protocol publication

[…] Isolation of epithelial tissue from the different parts of the intestine was performed as previously described [, ], and genomic DNA was extracted with DNAeasy blood and tissue kit (Qiagen). DNA concentration was assessed using a Nanodrop 2000 spectrophotometer (Thermo Scientific). For optimal results, 10ng of DNA per PCR reaction was used. QPCR was performed in a StepOne Plus Real-Time PCR System (Applied Biosystems) using iQ SYBR Green Supermix (Bio-Rad) and specific primers. Primers were designed by Primer3 software and tested with NCBI Primer Blast. Two sets of primers were designed for each gene; one set amplified in the presence of cre-mediated recombination and another one in its absence. 10ul reactions were performed in duplicate and standardized using relative amount of intron 1 product of the Tgfβ-RII gene as the internal control. Melting curve analysis was performed with each run to test specificity in SYBR green assays. Percentage of recombination was calculated by establishing the ratio between recombinant and non-recombinant alleles for each mouse. […]

Pipeline specifications

Software tools Primer3, Primer-BLAST
Application qPCR
Organisms Mus musculus, Homo sapiens
Diseases Colorectal Neoplasms, Hereditary Nonpolyposis, Leigh Disease, Neoplasms, Neoplastic Syndromes, Hereditary
Chemicals Aspirin