New insights into the expression profile and function of micro-ribonucleic acid in human spermatozoa Albert Salas-Huetos, M.Sc., Joan Blanco, Ph.D.,

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New insights into the expression profile and function of micro-ribonucleic acid in human spermatozoa Albert Salas-Huetos, M.Sc., Joan Blanco, Ph.D., Francesca Vidal, Ph.D., Josep M. Mercader, Ph.D., Nicolás Garrido, Ph.D., Ester Anton, Ph.D. Fertility and Sterility Volume 102, Issue 1, Pages 213-222.e4 (July 2014) DOI: 10.1016/j.fertnstert.2014.03.040 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) miRNA Ct expression values classified as Determined, Undetermined, or Unreliable. The miRNAs fraction of each category present or absent in all individuals has been indicated by stripes. (B) Hierarchical clustering of the 10 individuals based on the miRNA Ct values obtained. Samples are represented by column (n = 10) and assays by row (n = 768). Fertility and Sterility 2014 102, 213-222.e4DOI: (10.1016/j.fertnstert.2014.03.040) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Electrophoretic gel of PCR products corresponding to (A) PRM1 amplification. Ladder: molecular marker (0.1–1 kb). C+ = Genomic DNA as a positive control (422b). S01-S10 = Sperm cDNAs showing an intronless product obtained from RT (331b). C− = negative controls (RT-PCR without RNA and PRM1 PCR without cDNA). (B) GAPDH amplification. Ladder: molecular marker (0.1–1kb). C+ = Genomic DNA as a positive control (332b). S01-S10 = Sperm cDNAs showing an intronless product obtained from RT (228b). C− = negative controls (RT-PCR without RNA and GAPDH PCR without cDNA). (C) CD45 amplification. Ladder: molecular marker (0.1–1kb). C+ = Complementary DNA obtained from peripheral blood lymphocytes as a positive control (179b). S01-S10 = Sperm cDNAs showing the absence of CD45 marker. C− = negative controls (RT-PCR without RNA and CD45 PCR without cDNA). Fertility and Sterility 2014 102, 213-222.e4DOI: (10.1016/j.fertnstert.2014.03.040) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Electrophoretic analysis results of the sperm RNA samples using the Agilent 2100 Bioanalyzer. (A) Small-RNA chip (RNAs ranging from 4 to 150 nt). (B) Nano-RNA chip (RNAs ranging from 25 to 200 nt). The last electropherogram (in A and B) was obtained from Jurkatt cells RNA (somatic cell control). Fertility and Sterility 2014 102, 213-222.e4DOI: (10.1016/j.fertnstert.2014.03.040) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Results of the Spearman correlations performed between samples regarding the ubiquitous miRNA normCt expression values. Fertility and Sterility 2014 102, 213-222.e4DOI: (10.1016/j.fertnstert.2014.03.040) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 4 Heatmap showing the relative similitude between samples according to miRNA normCt values. Fertility and Sterility 2014 102, 213-222.e4DOI: (10.1016/j.fertnstert.2014.03.040) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions