Open Access Highly Accessed Research article

miRNAs associated with chemo-sensitivity in cell lines and in advanced bladder cancer

Iver Nordentoft1*, Karin Birkenkamp-Demtroder1, Mads Agerbæk2, Dan Theodorescu3, Marie Stampe Ostenfeld1, Arndt Hartmann4, Michael Borre5, Torben F Ørntoft1 and Lars Dyrskjøt1

Author Affiliations

1 Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark

2 Department of Oncology, Aarhus University Hospital, Aarhus, Denmark

3 University of Colorado Comprehensive Cancer Center, Aurora, CO 80045, USA

4 Department of Pathology, University of Erlangen, Erlangen, Germany

5 Department of Urology, Aarhus University Hospital, Aarhus, Denmark

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BMC Medical Genomics 2012, 5:40  doi:10.1186/1755-8794-5-40

Published: 6 September 2012

Additional files

Additional file 1:

Table S1. Response cohort - patient characteristics and treatment regimens.

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Additional file 2:

Table S2. Survival cohort - patient characteristics and treatment regimens.

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Additional file 3:

Table S3. miRNA normalization. Normalization of miRNA expression is challenging and therefore we chose to make 4 different normalizations prior to analysis of differentially expressed miRNAs (PD vs.CR) and compare the results. Normalizes used were mammalian U6 (MammU6) and RNU48 RNAs that is often used as normalizes, miR-193b selected using the Norm Finder program, and finally quintile normalization using the qpcrNorm program (Mar J et al. Data-driven Normalization Strategies for qPCR Data. Technical Report, 2008). The results show a large degree of agreement between normalizations. We argue that the Norm Finder approach is the best normalize approach for our data because quintile normalization is better suited for large gene sets and miRNA-193b is more stable than MammU6) and RNU48 across the samples (data not shown).

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Additional file 4:

Table S4. Technical validation of miRNA profiling. As a technical evaluation of the Taqman Human Array MicroRNA Cards (LDA analysis) the 6 top ranked miRNAs from the Array analysis were determined using real-time q-RT-PCR (n = 3) (singleplex) and compared to the MicroRNA card analysis. miR-193b expression was used as normalize.

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Additional file 5:

Table S5. miRNA normalization. Tree different normalizations prior to analysis of differentially expressed miRNAs (SS vs.LS) were performed to inspect the robustness of the analysis. Normalizes used were mammalian U6 (MammU6), miR-324-3p selected using the Norm Finder program and miR-193b. The results show a large degree of agreement between normalizations.

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Additional file 6:

Figure S1. miRNA down-regulation in bladder cell lines. LNA based antagonism were used to silence miR-27a and miR-138 in the bladder cell lines 253JBV and CLR2169. LNA knockdown molecules were reverse Transfected (n = 3) and after 48 h incubation culture media, with or without cisplatin (GI50), was added to the cells (n = 4). Expression of mature miR-27a and miR-138 was determined using real-time Q-PCR (n = 3). LF2000 designate controls using the transfection reagent alone. The 2-ΔΔCT method was used for relative quantification with miR-193b expression normalize.

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