miRNA array hybridization and analysis
The small RNAs were prepared from human prostate tumor and normal specimens using mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA). The N7 position of guanine of isolated small RNA nucleic acids were labeled with Cy3 using the ULS™ Small RNA Labeling Kit according to the manufacturer’s instructions (Kreatech Biotechnology, Amsterdam, The Netherlands). The labeled samples were incubated at 85°C for 15 minutes, and then spun down to collect the contents of tube before proceeding with purification using the KREApure columns (Kreatech Biotechnology, Amsterdam, The Netherlands). ULS-labeled small RNAs were purified by a KREApure, and stored at -70°C, or used for miRNA array hybridization. MiRNA arrays were generated on glass slides using the mirVana miRNA Array Probe Set (Ambion, Austin, TX, USA). The 210 mature miRNA complementary oligonucleotides were assembled and integrated into our miCHIP. Each probe was printed in duplicates. After hybridization, the miRNA arrays were scanned and analyzed using a GenePix 4000A array scanner (Axon Instruments Union City, CA, USA). Normalization was performed by expressing each miRNA replicate relative to a control miRNA; this process allowed us to make comparisons between chips.
Supplementary Figure Legends
Supplementary Figure 1:miRNAs differentially expressed in prostate normal and tumor samples. MiRNA extracting and profiling was performed as described in the Materials and Methods section by hybridization to miRNA-specific probes. The samples from prostatetumor tissue miRNAs and miRNAs obtained from adjacent non-tumor tissue were labeled with cys-3 subjected for the miCHIP analysis. Representative chip images and spots for selected miRNA that was decreased expression in prostate tumor tissue.
Supplementary Figure 2:The three NT paired tissues were used for miR-330 and E2F1 expression examination. The tumors were with lower levels of miR-330 than their adjacent non-tumor tissueshad higher levels of E2F1 protein than the non-tumor tissues.
Supplementary Figure 3:MiR-330 down-regulates E2F1 in prostate cancer cells. DU145 and 22Rv1 cell lines were transiently transfected with pre-miR-330, or a control-miRNA, respectively.MiR-quantitative RT-PCR and immunoblotting experiments were performed to detect the matured miR-330 level and protein levels of E2F1.
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