Abi prism 7500 sequence analyzer

Total cellular RNA was isolated from cell lines or transfected cells (RNA isolation Kit II, Macherey-Nagel, Düren, Germany; RNeasy micro Kit; Qiagen, Hilden, Germany) and reversely transcribed to cDNA using Superscript II and random hexamer primers (both Life Technologies GmbH, Darmstadt, Germany) or QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Quantitative real-time RT-PCR (qPCR) was performed in an ABI Prism 7500 Sequence Analyzer (Applied Biosystems, Foster City, CA) using 200 ng of cDNA and SensiMix^™II Probe Kit (Bioline GmbH, Luckenwalde, Germany) according to the manufacturer’s instructions. Primers and FAM (6-carboxyfluorescein)/TAMRA (tetramethylrhodamine)-labeled probes for detection of β-actin (ACTB) transcripts and 4-1BB have been described before [[70 (link)]]. For quantitation of Fascin transcripts, a TaqMan Gene Expression Assay (Hs00979631_g1; Applied Biosystems) was used. Expression levels were computed by interpolation from standard curves generated from plasmids carrying the respective target sequences and calculating the mean of triplicate samples. Each sample was measured in at least three biological replicates. ACTB was used for normalization.

10⁷ Jurkat T-cells were transfected with 50μg pEFTax or pEF. 5x10⁵ 293T cells were transfected with 2μg of pEFTax or pEF (see Transfections). 48h later, total cellular RNA was isolated from transfected Jurkat or 293T cells (RNA isolation Kit II, Macherey-Nagel, Düren, Germany) and reversely transcribed to cDNA using SuperScript II and random hexamer primers (both Life Technologies GmbH). 200ng of cDNA and SensiMix II Probe Kit (BioLine GmbH, Luckenwalde, Germany) were used according to the manufacturer’s instructions for quantitative real-time RT-PCR (qPCR) in an ABI Prism 7500 Sequence Analyzer (Applied Biosystems, Foster City, CA, USA). Primers and FAM (6-carboxyfluorescein) / TAMRA (tetramethylrhodamine)-labeled probes for detection of β-actin (ACTB) and Tax transcripts have been described before [46 (link)]. A TaqMan Gene Expression Assay (Hs00979631_g1; Applied Biosystems) was used for quantitation of Fascin transcripts. Expression levels were computed by interpolation from standard curves generated from plasmids carrying the respective target sequences and calculation of the mean of triplicated samples. Relative copy numbers (rcn) were determined by normalizing copy numbers on those of ß-actin (ACTB). At least, three independent experiments were performed.

At 48 h after transfection or inhibitor treatment, total cellular RNA was extracted (NucleoSpin® RNA, Macherey Nagel, Düren, Germany) and reversely transcribed to cDNA using random hexamer primers and Superscript™ II Reverse Transcriptase (both Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturers’ instructions. 200 ng of cDNA was subjected to quantitative real-time RT-PCR (qPCR) applying TaqMan® Universal PCR Master Mix and an ABI Prism 7500 Sequence Analyzer (both Applied Biosystems, Foster City, CA, United States) according to the manufacturers’ instructions. Primer sequences and FAM (6-carboxyfluorescein)/TAMRA (tetramethylrhodamine)-labeled probes for detection of β-actin (ACTB) and Tax transcripts have been described before [99 (link)]. Tax transcripts were additionally detected using the probe 5′-FAM-TACAAGGCGACTGGTGCC-TAMRA-3′. Standard curves were generated for Tax by pEF-Tax1 expression plasmid or for ACTB by pJET1.2/blunt plasmid (Fermentas, St.-Leon Roth, Germany) bearing a respective ACTB target sequence to compute transcript expression levels. The mean of technical triplicates was calculated for all samples. Every experiment was independently performed at least three times and relative copy numbers (rcn) were calculated by normalization of respective transcript levels on those of β-actin.