Comprehensive Molecular Characterization of ccRCC

DNA samples from ccRCC patients tumour and matching normal were all obtained under local IRB and LREC approvals for this study and processed as previously described2 (link). DNA fragmentation, library preparation and solution phase hybrid capture were according to manufacturer instructions (Agilent Technologies, US) and modified from previously published protocols5 . Capillary-based Sanger sequencing for confirmations and PBRM1 followup were done as previously described2 (link) with manual inspection of all sequencing traces. mRNA was extracted from snap-frozen mouse pancreatic lesions and subjected to RT-PCR using a nested PCR approach utilising primers of mouse Pbrm1 exon 23/24 and the Carp-β-Actin Splice acceptor sequence of the T2Onc transposon cassette. Resulting bands were gel-purified and subjected to capillary-based Sanger sequencing. PBRM1 or scrambled control siRNAs (Santa Cruz, CA) were transfected into ccRCC cell lines using Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer's conditions. Real-time PCR and western blotting were all done utilising standard protocols essentially as described1 (link). Expression analyses were carried out as previously described2 (link).

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Varela I., Tarpey P., Raine K., Huang D., Ong C.K., Stephens P., Davies H., Jones D., Lin M.L., Teague J., Bignell G., Butler A., Cho J., Dalgliesh G.L., Galappaththige D., Greenman C., Hardy C., Jia M., Latimer C., Lau K.W., Marshall J., McLaren S., Menzies A., Mudie L., Stebbings L., Largaespada D.A., Wessels L.F., Richard S., Kahnoski R.J., Anema J., Tuveson D.A., Perez-Mancera P.A., Mustonen V., Fischer A., Adams D.J., Rust A., Chan-on W., Subimerb C., Dykema K., Furge K., Campbell P.J., Teh B.T., Stratton M.R, & Futreal P.A. (2011). Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature, 469(7331), 539-542.

Publication 2010

Actin Capillary Carp Cell lines Dna fragmentation Exon Frozen Hybrid Library Lipofectamine 2000 Mouse Mrna Nested pcr Patients Pbrm1 Primers Real time pcr Rt pcr Sirnas Transposon Tumour dna

Corresponding Organization :

Other organizations : Wellcome Sanger Institute, National Cancer Centre Japan, University of Minnesota, The Netherlands Cancer Institute, Université Paris-Sud, Institut Gustave Roussy, Spectrum Health, Cancer Research UK, University of Cologne, Van Andel Institute, National University of Singapore, Genomic (Brazil)

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Variable analysis

independent variables

PBRM1 or scrambled control siRNAs (Santa Cruz, CA) were transfected into ccRCC cell lines using Lipofectamine 2000 (Invitrogen, CA)

dependent variables

Real-time PCR and western blotting
Expression analyses

control variables

DNA samples from ccRCC patients tumour and matching normal were all obtained under local IRB and LREC approvals
DNA fragmentation, library preparation and solution phase hybrid capture were according to manufacturer instructions (Agilent Technologies, US) and modified from previously published protocols
Capillary-based Sanger sequencing for confirmations and PBRM1 followup were done as previously described
MRNA was extracted from snap-frozen mouse pancreatic lesions and subjected to RT-PCR using a nested PCR approach utilising primers of mouse Pbrm1 exon 23/24 and the Carp-β-Actin Splice acceptor sequence of the T2Onc transposon cassette

positive controls

Sanger sequencing for confirmations and PBRM1 followup

negative controls

Scrambled control siRNAs

Annotations

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