Oligo d t 16 primer

RNA was isolated at 1, 2.5, and 6 days post-transfection of early region plasmid DNA using the Macherey & Nagel Total RNA Isolation Kit (Macherey & Nagel, Düren, Germany) according to the manufacturer´s instruction. DNA was removed by an additional Turbo DNA-free™ DNase treatment (Ambion, Austin TX, USA). RNA concentrations were determined with the NanoDrop™ 8000 (ThermoScientific, Waltham MA, USA) at 260 nm. Synthesis of cDNA was carried out with 500 ng RNA using SuperScript® II Reverse Transcriptase and Oligo(dT)16 primers (Invitrogen, Carlsbad CA, USA).

Total RNA was isolated, cDNA prepared with oligo d(T)16 primers (Thermo Fisher Scientific), and quantitative real-time PCR (qPCR) reactions performed with 1/10 diluted cDNA templates with the Fast SYBR Green Master Mix (Thermo Fisher Scientific) or the TaqMan Fast Universal Master Mix (Thermo Fisher Scientific) as described previously (19 (link)). Relative gene expression was calculated using the 2^-ΔCt method and the reference genes β-actin and/or Hprt were used to normalize gene expression. Primers and probes were obtained from Integrated DNA Technologies and sequences are available upon request.

Total RNA was isolated using QIAzol Lysis Reagent and miRNeasy Mini Kit (QIAGEN, Hilden, Germany) with DNase I digestion (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. RNA integrity was checked through agarose gel electrophoresis. RNA yield and A260/280 ratio were monitored with a NanoDropND-2000c spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). For gene-expression profiling studies, RNA integrity numbers were also measured using 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Normal prostate tissue RNA used as calibrator for qRT-PCR analysis was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
cDNA was synthesized using high-capacity cDNA Reverse Transcription Kit with random primers (Thermo Fisher Scientific Inc., Waltham, MA, USA) for total RNA or oligo-dT₁₆ primers (Thermo Fisher Scientific Inc., Waltham, MA, USA) for nuclear and cytoplasmic fraction. For miRNA detection, TaqMan MicroRNA Reverse Transcription Kit and sequence specific primers (Thermo Fisher Scientific Inc., Waltham, MA, USA), listed in Supplementary Table 4, were used on total RNA.

Total RNA was extracted using an RNeasy Micro Kit (Qiagen). RNA concentration and purity was quantified on a NanoDrop. 1 μg of RNA was reverse transcribed using Oligo-d(T)₁₆ primers (Applied Biosystems), and M-MLV reverse transcriptase (Invitrogen). Gene expression was analyzed on an ABI Prism 7900HT Sequence Detector with SA Biosciences RT² Real-Time SYBR Green master mix (Qiagen). Quantitect Primer Assays (Qiagen) were used for HPRT (QT00059066), 18S (QT00248682), Dll1 (QT00057631), Nrp1 (QT00023009), VEGFR2 (QT00069818) and Notch1 (QT01005109). All other primers were designed in-house (Table 1). The data was normalized to two endogenous controls, HPRT and 18S, using the ΔΔCt method.

To prepare RNA for cDNA synthesis, 1–2 µg of RNA was diluted in diethyl pyrocarbonate-treated water (Thermo Fisher Scientific) and denatured for 5 min at 65°C to remove the secondary structure. The 20 µL reaction mixture also contained reverse transcriptase, (M-MLV RT, Promega, Madison, WI, UnitedStates), 4 mM dNTPs (Promega), 2.5 µM Oligo dT16 primers (Applied Biosystems, Waltham, MA, UnitedStates) and was incubated for 1 h at 37°C. Reverse transcription was terminated by incubation at 95°C for 5 min.
For PCR amplification, sense and anti-sense primers were designed using the Primer Express 2 software (Applied Biosystems). Oligonucleotides were purchased from Invitrogen (Table 2).