Gene expression assay

Real time TAQMAN RT-PCR assays were performed as described by García-Cruz et al.^{60 (link)}. Total RNA was extracted from about 1 × 10⁶ cells using TRIZOL reagent (Life Technologies, Inc.). cDNA was reverse-transcribed from total RNA samples using SuperScriptIII® from Invitrogen. The resulting cDNA was amplified by PCR using Taq Man Assay primers with the Taq Man Universal Non-amperase PCR Master Mix and analyzed with a 7500 ABI PRISM Sequence Detector System according to the manufacturer’s instructions. mRNA expression was calculated from the relevant signals by normalization with respect to the signal for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. The assay numbers for exons E3-IDX p19 H-RAS (Hs00978053_g1), and E4A-E4B H-RAS total (Hs00978051_g1) and for GAPDH (HS99999905_m1 housekeeping) were supplied by Applied Biosystems Gene Expression Assays (Applied Biosystems). Assays were run with Taqman Universal.

According to the manufacturer’s instruction, we extracted total RNA using Trizol Reagent (Ambion, Austin, TX, USA) and cDNAs with the Superscript Vilo kit (Invitrogen, Waltham, MA, USA). The reverse transcription reaction was performed at 42 °C for 30 min, at 99 °C for 5 min, and then cooled to 4 °C. We held the PCR mixture at 94 °C for 2 min and then cycled 30 times at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, followed by 10 min at 72 °C at the final cycle. We performed real-time PCR assays with TaqMan Universal PCR Master Mix and gene expression assays from Applied Biosystems (Waltham, MA, USA). We normalized gene expression with human gapdh as the endogenous control. We used the 2Δct method to analyze data and expressed data as arbitrary units or as fold difference.

Cell samples were collected using 0.25% trypsin-EDTA (Invitrogen) to remove the cells from cell culture plates. Total RNA was purified using QIAGEN RNeasy Mini kit. Possible genomic DNA contamination was removed by RNase-Free DNase Set (QIAGEN) with the RNeasy columns, or by DNase I (Invitrogen) treatment for 15 min at room temperature. A total of 500 ng of total RNA was used for Oligo(dT)₂₀ – primed reverse transcription using SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR was performed using Taqman PCR Master Mix and Gene Expression Assays (Applied Biosystems, Supplementary file 1b) in triplicate for each sample and each gene. A total of 0.5 μl of cDNA from RT reaction was added as template for each Q-PCR reaction. The expression of genes of interest was normalized to that of GAPDH. RT-PCR was carried out using Platinum Taq DNA Polymerase (Invitrogen) or Gotaq Master Mix (Promega) and then subjected to 2% agarose gel electrophoresis. PCR conditions included denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min, for 35 cycles, with 72 °C extension for 7 min at the end. ACTB (β-actin) was used as an endogenous control.

RNA was extracted in TRIzol and processed in a Real-time PCR equipment as previously described^{7 ,32 (link)}. Samples were analyzed in duplicate vs. 36B4. Dio2, Ucp1 and Dio1 were assayed using specific Taqman probes (Mm01244861m1 for Ucp1, Mn00515664m1 for Dio2, Mn00839358_m1 for Dio1 (Gene expression assays, Applied Biosystems, Foster City, CA). Results were normalized to cyclophilin labelled with VIC (Ppia, Mm02342429g1, Applied Biosystems) in the same well. The fold-change in mRNA expression was calculated by the 2^−ΔΔCt method.

Total RNA was isolated using the RNeasyPlus Kit (Qiagen, Hilden, Germany) according to the manufacturer instruction. The quantity and purity of RNA was determined by measuring the optical density at 260 and 280 nm. 600 ng total RNA was reverse transcribed to cDNA with TaqMan Reverse Transcription Reagents (Applera GmbH, Darmstadt, Germany). For qRT-PCR the Fluidigm Biomark high throughput qPCR chip platform (Fluidigm Corporation, San Francisco, CA, USA) with pre-designed gene expression assays from Applied Biosystems was used according to the manufacturer instructions (Spurgeon et al., 2008 (link)). Data were analyzed using the ddCT method (Livak and Schmittgen, 2001 (link)) and expression values were normalized to the expression levels of the β-actin gene. All TaqMan assays are listed in the Supplementary Material in Data Sheets 1, 2 for stimulation with IL-1β/TNFα and BMDM supernatant, respectively.

Total RNA was isolated from cell lines and tumors using the SV Total RNA Isolation System (Promega, USA). A total of 1 μg of RNA was reverse transcribed to cDNA following M-MLV Reverse Transcriptase (Invitrogen, USA) and random hexamers (Amersham Pharmacia, UK) for reverse transcription. Analysis of hCNT1, hCNT2, hCNT3, hENT1, hENT2 and GAPDH (internal control) mRNA levels were performed by RT-PCR using TaqMan Gene Expression Assays (Applied Biosystems, USA) as previously described [44 (link)]. The mRNA expression of hOCT1 was assessed using the commercial Gene Expression Assays (Applied Biosystems). Relative quantification of gene expression was assessed using the ΔΔCT method, as described in the TaqMan user's manual (User Bulletin no. 2; Applied Biosystems). Gene expression levels for each individual sample were normalized relative to the GAPDH gene. The amounts of mRNA were expressed as arbitrary units.
Absolute quantification of gene expression was performed by using DNA plasmids containing each of the analyzed transporters to construct standard curves based on serial dilutions of the plasmids. The standard curves allowed us to correlate CT values of the samples with the mRNA copy number of each gene per microgram of total RNA.