User bulletin no 2

For measuring the relative mRNA expression of target genes, total RNA from esophagus and tongue from WT and IL-1β mice (n = 10–13 per group, SPF only) was extracted using Trizol reagent following manufacturer’s instructions (Invitrogen, Carlsbad, CA). Specifically, 2 µg (esophagus) or 4 µg (tongue) of the total RNA from each sample was reverse transcribed into cDNA using the High Capacity cDNA Archive kit (Life Technologies). Levels of Il-6, Tnf-α, Inf-γ, Il-33, Il-17A, Foxp3 + and iNos mRNA in the esophageal and tongue tissues were measured by qPCR using commercial primers and probes (TaqMan Gene Expression assays) in the 7500 FAST real-time PCR system. The mRNA levels were normalized to the endogenous control glyceroldehyde-3-phosphate dehydrogenase mRNA (Gapdh) and expressed as fold change in reference to wild type littermates using the comparative threshold cycle (C_T) method (Applied Biosystems User Bulletin No.2). Human IL-1β expression was determined by semi-quantitative PCR (Supplemental Fig. S1) using forward (5ʹ-ACCTCCAGGGACAGGATATGG-3ʹ) and reverse (5ʹ-CTCCAGCTGTAGAGTGGGCTTAT-3ʹ) primers with PCR condition 95 °C for 3 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and 72 °C for 5 min.

Mononuclear cells were obtained by Ficoll-Paque gradient centrifugation. Total RNA was extracted using Trizol reagent (Life Technologies) according to the manufacturer’s instructions. Reverse transcription was performed using 1 μg of total RNA from each sample by High Capacity cDNA Reverse Transcription Kit (Applied Biosistem, Foster City, CA, USA). qRT-PCR was performed as described previously.^{57 (link)} Simultaneous quantification of ABL1 mRNA was used as a reference for mRNA TaqMan assay data normalization. miR-128 expression was normalized on RNU44. The comparative cycle threshold (Ct) method for relative quantification of mRNA and miRNA expression (User Bulletin No. 2; Applied Biosystems) was used to determine transcript levels.

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.