7900ht real time pcr cycler

Total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol including a DNase digestion step. The concentration and quality of the RNA was determined with a Nano Drop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). 2 µg of RNA were reverse transcribed by 200 U Moloney murine leukemia virus reverse transcriptase (Promega, Heidelberg, Germany) using 100 pmol of oligo (dT)₁₅ as primers (2 h at 42 °C). Real-time quantitative PCR was performed on a 7900HT real-time PCR cycler (Applied Biosystems, Darmstadt, Germany). Experiments were set up in triplicates containing SYBR Green Mastermix (AbGene, Hamburg, Germany) and the respective primers (3 pmol per reaction). The primer sequences are available from Table 1(Tab. 1). The thermal cycling protocol comprised an initial denaturation step at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 1 min, and a final extension at 60 °C for 15 min. The mRNA amount of CYP1A1 in each sample was normalized to its ACTB (encoding β-actin) content and then referred to vehicle controls treated with the solvent DMSO only to obtain fold inductions.

Snap-frozen tissues or cells were homogenized directly into Trizol reagent for RNA analysis or lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA with protease inhibitors [Complete Mini, Roche]) for protein work. mRNA was extracted using RNeasy kits (Qiagen) and normalized for cDNA synthesis, and expression was assessed by quantitative RT-PCR using TaqMan reagents on a 7900HT Real Time PCR cycler (all Applied Biosystems). A complete list of probes used for quantitative RT-PCR is available in Supplemental Table 2. Protein expression was analyzed by Western blotting from clarified lysates normalized for protein content by BCA method (Bio-Rad), all as previously described (46 (link)). A complete list of primary antibodies can be found in the Reagents section.