Open Access Highly Accessed Research article

Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor

Franc Llorens12345, Manuela Hummel12, Lorena Pantano1267, Xavier Pastor127, Ana Vivancos128, Ester Castillo12, Heidi Mattlin129, Anna Ferrer12, Matthew Ingham129, Marc Noguera107, Robert Kofler111122, Juliane C Dohm1112, Raquel Pluvinet7, Mònica Bayés129, Heinz Himmelbauer12, José Antonio del Rio345, Eulàlia Martí126 and Lauro Sumoy127*

Author Affiliations

1 Centre for Genomic Regulation (CRG), Barcelona, Spain

2 Universitat Pompeu Fabra (UPF), Barcelona, Spain

3 Molecular and Cellular Neurobiotechnology Group, Institut de Bioenginyeria de Catalunya (IBEC), Barcelona, Spain

4 Department of Cell Biology, University of Barcelona (UB), Barcelona, Spain

5 Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain

6 Centro de Investigación Biomédica en Red en Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain

7 Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Spain

8 Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Spain

9 Centro Nacional de Análisis Genómicos (CNAG), Barcelona, Spain

10 Molecular Epidemiology Group, IRSI-Caixa Foundation, Barcelona, Spain

11 Max Planck Institute for Molecular Genetics, Berlin, Germany

12 Institute for Population Genetics, University of Veterinary Medicine, Vienna, Austria

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BMC Genomics 2013, 14:371  doi:10.1186/1471-2164-14-371

Published: 1 June 2013

Additional files

Additional file 1:

EGF activation of the MAPK pathway in HeLa cells. Serum-starved HeLa cells were stimulated with EGF at the indicated times. Total cell extracts were prepared as indicated in Materials and Methods and samples were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.

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Additional file 2:

Intensity correlation between replicate samples processed on Exiqon microarrays. (A,B) Correlation of normalized log2intensities between biological replicates in the Control (A) and the EGF group (B) measured on Exiqon microarrays.

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Additional file 3:

Intensity correlation between replicate samples processed on Agilent microarrays. (A,B) Correlation of normalized log2intensities between biological replicates in the Control (A) and the EGF group (B) measured on Agilent microarrays. Sample 1 was processed with two technical replicates ('1' and '1_b').

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Additional file 4:

Summary of read counts generated by the miRNA-seq MIRO analysis pipeline.

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Additional file 5:

Read count correlation between replicate samples processed by Illumina sequencing. (A,B) Correlation of normalized, log2transformed read counts per miRNA between biological replicates in the Control (A) and the EGF group (B) measured by Illumina small RNA-seq.

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Open Data

Additional file 6:

Numbers of miRNAs present in the different platforms. The Venn diagram shows how many miRNAs are present on the two microarray platforms, how many were detected by Illumina sequencing, and the numbers of the respective overlaps.

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Additional file 7:

Agilent, Exiqon and Illumina EGF versus control log2ratios, fold changes, statistical significance nominal and adjusted p-values, and ranks computed with Rankprod.

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Additional file 8:

Inhibition of the MAPK pathway in HeLa cells. HeLa cells were serum-starved for 24 hours and treated with EGF for 6 hours in the presence or absence of protein kinase inhibitors: AG1470 (EGFR inhibitor), U0126 (MEK inhibitor) and Wortmannin (PI3K inhibitor). In addition, HeLa cells were transfected with a constitutively active form of Ras (RasV12). Lysates were analysed by Western-Blot against pospho-ERK1/2 and phospho-AKT to ensure protein inhibitors and transfection action over MAPK and AKT pathways. Tubulin was used as sample loading control.

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Additional file 9:

List of 234 genes predicted in common by TS5.0 miRvana 3.0 and PICTar to be targets of the 8 miRNAs found to be regulated by EGF in this work.

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Additional file 10:

Summary of isomiR counts for top EGF regulated miRNAs.

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Additional file 11:

Listing of isomiR sequence variants and their respective read counts for top EGF regulated miRNAs.

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