Research article
Characterisation of microRNA expression in post-natal mouse mammary gland development
-
* Corresponding authors: Eric A Miska e.miska@gurdon.cam.ac.uk - Carlos Caldas carlos.caldas@cancer.org.uk
- † Equal contributors
1 Breast Cancer Functional Genomics Laboratory, Cancer Research UK Cambridge Research Institute and Department of Oncology, University of Cambridge, Li Ka Shing Centre, Cambridge, UK
2 Computational Biology Group, Cancer Research UK Cambridge Research Institute and Department of Oncology, University of Cambridge, Li Ka Shing Centre, Cambridge, UK
3 Mammary Stem Cell Biology Group, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge, UK
4 Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK
5 Computational Biology Group, Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge, UK
6 Department of Pathology, University of Cambridge, Cambridge, UK
7 Current address: Department of Pathology, Technische Universität München, Munich, Germany
BMC Genomics 2009, 10:548 doi:10.1186/1471-2164-10-548
Published: 20 November 2009Additional files
Additional file 1:
Cellular composition of mouse mammary glands. Changes in the cellular composition of the mammary gland are shown for eight developmental time points up to mid-gestation. The proportion of luminal, basal, and stromal cells was measured by fluorescence-activated cell sorting (FACS) of dissociated total mouse mammary glands.
Format: PDF Size: 104KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 2:
Validation of gene (mRNA) expression by qRT-PCR. a. Validation of selected mRNA genes by qRT-PCR using custom designed primers and SYBR Green reporter (left panel), normalising data to the expression values of housekeeping genes beta-actin (Actb) and cyclophilin A (Ppia) (bottom rows). Expression data obtained by Illumina profiling (middle panel) showed good correlation with qRT-PCR data (right panel). b. Detailed description of SYBR Green qPCR according to MIQE guidelines.
Format: PDF Size: 829KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 3:
Hierarchical clustering of time points based on mRNA expression data. Prior to clustering log2 intensities were mean centred across time points and multiple probes assigned to the same Entrez ID were summarized by their mean expression profile.
Format: PDF Size: 79KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 4:
Enrichment and depletion of GO biological processes (a, b) and KEGG pathways (c, d) among predicted miRNA targets. Target genes were defined as genes with at least one 3' UTR match to the seed sequence of one or more miRNAs in a given cluster (a, c) or to individual seed-identical miRNA families (b, d). Log2 fold-enrichments are shown as heatmaps with red and green corresponding to enrichment and depletion, respectively. Significance was assessed by a two-sided Fisher's Exact test. Shown are gene sets with Benjamini-Hochberg corrected P < 0.01 (out of all statistically significant GO terms for a given cluster or miRNA family, only the most specific GO terms were included).
Format: PDF Size: 377KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 5:
miRNA expression and genomic location. Heatmap as in main text Figure 3b with miRNAs in individual clusters reordered according to their genomic loci. Brackets in the right-hand margin indicate genomic clusters. A mature miRNA was assigned to a genomic cluster if its locus was situated within 50 kb of another cluster member (genomic clusters in distinct regions of the genome that shared a common mature miRNA were merged).
Format: PDF Size: 194KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 6:
Higher-magnification images of miRNA in-situ hybridisation. Higher-magnification images of in-situ hybridisation for miRNAs let-7b, miR-205, and miR-206, showing higher expression in the epithelial compared to the stromal cell compartment, and specificity for the luminal and basal epithelial cell layers for let-7b and miR-205, respectively. White scale bars in images for time point 25d indicate 100 microns.
Format: PNG Size: 4.6MB Download file
Additional file 7:
Changes in the expression of predicted miRNA targets. Negative Log10 transformed two-sided P-values (see Methods) are shown as heatmaps with red and green corresponding to evidence for increased and reduced relative expression levels, respectively. Target genes were defined as genes with at least one 3' UTR match to the seed sequence of one or more miRNAs in a given cluster (a) or to individual seed-identical miRNA families (b). miRNA families were ordered by the evidence for systematic changes in the expression of their targets (based on the minimum observed P-value). Asterisks indicate Benjamini-Hochberg corrected P < 0.05. (c) Greatest evidence for systematic changes in target expression was observed for miRNA families miR-29, miR-200bc/429 and let-7/miR-98/1961. Dashed lines indicate the threshold for Benjamini-Hochberg corrected P-values smaller than 0.05.
Format: PDF Size: 358KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 8:
Validation of miRNA expression by qRT-PCR. a. Validation of selected miRNA genes by qRT-PCR using TaqMan miRNA Assays (left panel). qRT-PCR measurements were normalised to U6 small nuclear RNA (U6sRNA; bottom row). Expression data obtained by Luminex bead-based profiling (middle panel) showed good correlation with qRT-PCR data (right panel). b. Detailed description of TaqMan qPCR according to MIQE guidelines.
Format: PDF Size: 386KB Download file
This file can be viewed with: Adobe Acrobat Reader