Reverse transcription system

Total RNA was isolated from liver according to the manufacturer's instructions (RNeasy® Mini Kit, Qiagen). RNA samples were incubated with DNase I (Qiagen) for 15 min at room temperature prior to reverse transcription. Liver RNA (1 μg) was reverse transcribed into complementary DNA using the Reverse Transcription System (Applied Biosystems). After 4-fold dilution, 5 μl was used as a template for real-time RT-PCR. Amplification was done for 40 cycles using Power SYBR Green PCR master mix kit (Applied Biosystems). Quantification of mRNA was performed using the ΔΔC_T method and normalized to GAPDH. Primer sequences are as follows: GAPDH (NM_008084), 5′-CTC ATG ACC ACA GTC CAT GCC A-3′, 5′-GGA TGA CCT TGC CCA CAG CCT T-3′; SAA1.1 (NM_011314), 5′- GCC ATG GAG GGT TTT TTT CAT TTA TTG-3′, 5′-TTG TCT CCA TCT TTC CAG CC-3′; SAA2.1 (NM_009117), 5′-GCC ATG GAG GGT TTT TTT CAT TTA TTG-3′, 5′-GAG CAT GGA AGT ATT TGT CTG AG-3′.

Total RNA was extracted from tissues using the RNeasy Lipid Tissue Mini Kit (Qiagen Science, Hilden, Germany). RNA was converted to cDNA using the Reverse Transcription System (Applied Biosystems, Carlsbad, CA), and quantitative gene expression was analyzed by real-time PCR (RT-qPCR) on a 7900HT Fast Real-Time PCR System using the TaqMan Gene Expression Assay (Applied Biosystems). Details on the probes are provided in Supplementary Table S1: Additional File 17. B2m was used as the housekeeping gene and the relative expression was calculated using the comparative Ct method (2-ΔΔCt).

Total RNA was extracted from cells using TRI Reagent (Molecular Research Center, Cincinati, OH, USA). Quantification was performed at 260 nm and purity was assessed by the OD260/OD280 ratio. For gene expression analysis, 1 μg RNA was reverse-transcribed with random primers using a Reverse Transcription System (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR (qRT-PCR) was conducted on a ProFlex PCR System using TaqMan Gene Expression Assays (Applied Biosystems) (Supplementary Table 1). Results were calculated using the comparative Ct method (2-ΔΔCt) normalized to the expression of the housekeeping gene 18S (Hs 03928985_g1) and expressed relative to the control condition set to 1. Two technical duplicates were performed for each biological replicate.

As previously described^{32 (link)}, total RNA from tissues was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen Science, Hilden, Germany) and converted to cDNA using the Reverse Transcription System (Applied Biosystems, Carlsbad, CA). Quantitative gene expression was analyzed by real-time PCR (RT-PCR) on a 7900HT Fast Real-Time PCR System using the TaqMan Gene Expression Assay (Applied Biosystems) (Supplementary Table S1 online). Fold differences were calculated using the comparative Ct method (2^−ΔΔCt), and were expressed relative to the expression of the housekeeping gene 18S.

Fat tissue samples were homogenized using a conventional battery-operated, handheld homogenizer and solubilized in Tris Reagent (Sigma, St. Louis, MO, USA). Total RNA was isolated from adipose tissue and cells using the miRNeasy Mini kit (Qiagen Science, Hilden, Germany). RNA quantity was measured at 260 nm, and purity was assessed by the OD260/OD280 ratio. One microgram of RNA was transcribed to cDNA with random primers using the Reverse Transcription System (Applied Biosystems, Foster City, CA, USA). Quantitative gene expression was evaluated by real-time polymerase chain reaction (qPCR) on a 7900HT Fast Real-Time PCR System, using 384-well microfluidic TaqMan low-density array cards with an inventoried panel of gene expression assays from Applied Biosystems (Carlsbad, CA, USA). We performed an extensive literature search to assemble the mesothelial and mesenchymal expression profiles with well-established markers (Supplementary Table S2). Relative expression levels were calculated using the comparative Ct method and normalized to the expression of the housekeeping genes cyclophilin 1A (PPIA; reference gene for the tissue samples) and 18S (reference gene for ASCs samples).