Computational protocol: Genomic Landscape of Experimental Bladder Cancer in Rodents and Its Application to Human Bladder Cancer: Gene Amplification and Potential Overexpression of Cyp2a5/CYP2A6 Are Associated with the Invasive Phenotype

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Protocol publication

[…] Genomic DNA extracted from mouse tissues and paraffin-embedded human primary bladder tumors was analyzed by qPCR using SYBR Green PCR Master Mix and the 7300 or 7500 real-time PCR systems (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. PCR primers for mouse and human genes were designed using the Primer 3 software (www.broadinstitute.org/cgi-bin/primer/primer3_www.cgi) or the Primer Express Version 3.0 software (Applied Biosystems) and evaluated for specificity using in-silico PCR search (). Standard curves for mouse and human genes were constructed based on genomic DNA extracted from normal mouse liver and commercially available human (male) genomic DNA (product #G147A 26003101; Promega, Madison, WI, USA), respectively. Copy numbers of mouse and human genes were normalized to those of the mouse L1td1 and human LINE1 sequences, respectively, used as internal controls, and the copy number change was then calculated based on the assumption that normal mouse liver and human genomic DNA is diploid.The results were validated using genomic DNA extracted from cultured human cells. Gene copy numbers were estimated by qPCR using FAM-labeled probes (Universal Probe Library Set, Human Probe#1–90; Hoffmann-La Roche Ltd, Basel, Switzerland) and TaqMan Gene Expression Master Mix (Applied Biosystems). Primers and probes were designed using the website (https://qpcr.probefinder.com/input.jsp?organism=h_sap), and candidate sequences were examined for homology with other genomic regions and for the presence of known polymorphisms registered in dbSNP (). The candidate primers and probes were tested using germline DNA of a healthy woman, and primer-probe combinations were selected based on a standard titration curve with a slope of < -3 and R2 > 0.996. RNase P (Applied Biosystems) was used as an internal control.qPCR was performed in quadruplicate using an ABI7900HT instrument (Applied Biosystems). Each well contained FAM- and VIC-labeled probes for target genes and RNaseP, respectively, and the related primers. The threshold cycle (Ct) of the FAM probe was adjusted to that of the VIC probe, and ΔCt was calculated. Copy number was estimated according to the ΔCt of human genomic DNA, which was assumed to have two copies of respective genes. […]

Pipeline specifications

Software tools Primer3, Primer Express
Application qPCR
Organisms Mus musculus, Rattus norvegicus, Homo sapiens
Diseases Urinary Bladder Neoplasms, Neoplasms
Chemicals Butylhydroxybutylnitrosamine