American Society of Hematology

Figure 2

$Figure 2. Real-time quantification of mature miRNA in HTLV-I cell lines and ex vivo ATL samples. (A-D) TaqMan real-time RT-PCR from HTLV-I–transformed cells (C8166, MT2, MT4, and HUT102) and immortalized cells (LAF and MU04) in vitro and ATL tumor cells ex vivo. The TaqMan probes that were used ensure accurate discrimination between miRNAs that may differ by a single nucleotide. Mature miRNAs were analyzed because maturation of pri-miRNA is subject to posttranscriptional regulations. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. TaqMan CT values are used to measure the fold change. ATL samples and HTLV-I cell lines were compared with peripheral blood mononuclear cells (PBMCs) and purified CD4 T cells as controls. miR-24 was used as the internal control, because of its constitutive expression among ATL samples, control T cells, and HTLV-I cell lines. These experiments were performed by Asuragen (Austin, TX). All primers and probes were tested and validated by Asuragen. Statistical analysis is provided in Document S1. For panel D, MT4 cells were treated overnight with either 6 μM parthenolide (PTL) or 90 μM JNK II. Control cells were treated with DMSO. SYBR green real-time PCR was performed for pre-miR-155 expression (F: 5′-CTGTTAATGCTAATCGTGATAG-3′ and R: 5′-AATGCTAATATGTAGGAGTCAG-3′) with GAPDH (F: 5′-GAAGGTGAAGGTCGGAGTC-3′ and R: 5′-GAAGATGGTGATGGGATTTC-3′) as an internal control. All experiments were performed in triplicate.$

Real-time quantification of mature miRNA in HTLV-I cell lines and ex vivo ATL samples. (A-D) TaqMan real-time RT-PCR from HTLV-I–transformed cells (C8166, MT2, MT4, and HUT102) and immortalized cells (LAF and MU04) in vitro and ATL tumor cells ex vivo. The TaqMan probes that were used ensure accurate discrimination between miRNAs that may differ by a single nucleotide. Mature miRNAs were analyzed because maturation of pri-miRNA is subject to posttranscriptional regulations. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. TaqMan CT values are used to measure the fold change. ATL samples and HTLV-I cell lines were compared with peripheral blood mononuclear cells (PBMCs) and purified CD4 T cells as controls. miR-24 was used as the internal control, because of its constitutive expression among ATL samples, control T cells, and HTLV-I cell lines. These experiments were performed by Asuragen (Austin, TX). All primers and probes were tested and validated by Asuragen. Statistical analysis is provided in Document S1. For panel D, MT4 cells were treated overnight with either 6 μM parthenolide (PTL) or 90 μM JNK II. Control cells were treated with DMSO. SYBR green real-time PCR was performed for pre-miR-155 expression (F: 5′-CTGTTAATGCTAATCGTGATAG-3′ and R: 5′-AATGCTAATATGTAGGAGTCAG-3′) with GAPDH (F: 5′-GAAGGTGAAGGTCGGAGTC-3′ and R: 5′-GAAGATGGTGATGGGATTTC-3′) as an internal control. All experiments were performed in triplicate.

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