Next Generation Sequencing of Exosomal and Cellular miRNAs Between hCEp and Siha
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ABSTRACT:
PROVIDER: PRJNA600161 | ENA |
REPOSITORIES: ENA
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Project description:To investigate the differences in miRNA profiles, exosomal and cellular miRNA profiles between hCEp (Human Cervical Epithelia Cells) and Siha cells were generated by deep sequencing, in triplicate, using Illumina GAIIx. qRT–PCR validation was performed using TaqMan and SYBR Green assays
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Project description:Recently, the existence of extracellular miRNAs enclosed in exosomes has raised the possibility that they play an important role in cell-cell communication. To gain more insight into cell-cell communication via exosomal miRNAs, we investigated whether or not tumor cells exposed to hypoxia secrete exosomes which may affect angiogeneic activity. We used SUDHL4 cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from SUDHL4 cells cultured in normoxia (20%) or hypoxia (1%) for 24 h were used for validation of angiogeneic activity, such as tube formation assay. The exosome secreted from SUDHL4 cells in hypoxic condition significantly enhanced tube formation by HUVECs when compared with exosome obtained from SUDHL4 cell in normoxic condition. To identify cellular and exosomal miRNAs universally responding to hypoxic condition, we assess the expression profiles of intercellular and extracellular miRNAs in SUDHL4 cells cultured in normoxia (20%) or hypoxia (1%) for 24 h using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). SUDHL4 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).
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Project description:Recently, the existence of extracellular miRNAs enclosed in exosomes has raised the possibility that they play an important role in cell-cell communication. To gain more insight into cell-cell communication via exosomal miRNAs, we investigated whether or not tumor cells exposed to hypoxia secrete exosomes which may affect angiogeneic activity. We used RPMI8226 cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from RPMI8226 cells cultured in normoxia (20%) or hypoxia (1%) for 24 h were used for validation of angiogeneic activity, such as tube formation assay. The exosome secreted from RPMI8226 cells in hypoxic condition significantly enhanced tube formation by HUVECs when compared with exosome obtained from RPMI8226 cell in normoxic condition. To identify cellular and exosomal miRNAs universally responding to hypoxic condition, we assess the expression profiles of intercellular and extracellular miRNAs in RPMI8226 cells cultured in normoxia (20%) or hypoxia (1%) for 24 h using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). RPMI8226 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).
2013-12-03 | E-GEOD-45388 | biostudies-arrayexpress
Project description:Recently, the existence of extracellular miRNAs enclosed in exosomes has raised the possibility that they play an important role in cell-cell communication. To gain more insight into cell-cell communication via exosomal miRNAs, we investigated whether or not tumor cells exposed to hypoxia secrete exosomes which may affect angiogeneic activity. We used K562 cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from K562 cells cultured in normoxia (20%) or hypoxia (1%) for 24 h were used for validation of angiogeneic activity, such as tube formation assay. The exosome secreted from K562 cells in hypoxic condition significantly enhanced tube formation by HUVECs when compared with exosome obtained from K562 cell in normoxic condition. To identify cellular and exosomal miRNAs universally responding to hypoxic condition, we assess the expression profiles of intercellular and extracellular miRNAs in K562 cells cultured in normoxia (20%) or hypoxia (1%) for 24 h using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). K562 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).
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