18s rrna

RNA isolation and purification was performed as described earlier19 (link). Transcript levels for Aqp3 (NM_016689.2), Aqp7 (NM_007473.4), Aqp9 (NM_022026.2) and Pparg (NM_001127330.1) were quantified by real-time PCR (7300 Real Time PCR System, Applied Biosystems, Foster City, CA, USA). Primers and probes (Supplementary Table S1) were designed using the software Primer Express 2.0 (Applied Biosystems) and purchased from Genosys (Sigma). Primers or TaqMan^® probes encompassing fragments of the areas from the extremes of two exons were designed to ensure the detection of the corresponding transcript avoiding genomic DNA amplification. The cDNA was amplified at the following conditions: 95 °C for 10 min, followed by 45 cycles of 15 s at 95 °C and 1 min at 59 °C, using the TaqMan^® Universal PCR Master Mix (Applied Biosystems). The primer and probe concentrations were 300 and 200 nmol/L, respectively. All results were normalized for the expression of 18 S rRNA (Applied Biosystems), and relative quantification was calculated using the ΔΔCt formula19 (link). Relative mRNA expression was expressed as fold expression over the calibrator sample. All samples were run in triplicate and the average values were calculated.

Total RNA was isolated from renal biopsies using the High Pure RNA Isolation Kit and High Pure RNA Paraffin Kit (Roche Life Science, Mexico) according to the manufacturer's instructions. Single-stranded cDNA was synthesized by reverse transcription using the miScript Reverse Transcription Kit (Qiagen, United States) according to the manufacturer's instructions. Real-time PCR was performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with TaqMan gene expression assays. Comparative real-time PCR assays were performed for each sample in triplicate. The primers for WT1-F: 5′-TCTGCGGAGCCCAATACAG-3′; WT1-R: 5′-CACATCCTGAATGCCTCTGAAGA-3′; and WT1-P FAM: 5′-CACCGTGCGTGTGTATT-TAMRA-3′ were used. The comparative quantification cycle threshold (C_q) method was used to determine the relative expression levels of the WT1 gene. The 18S rRNA (Applied Biosystems, Foster City, CA, USA) gene was used for normalization.

For qPCR analyses, mRNA was isolated from FACS sorted CD4⁺ T-cell populations or from 16610D9 thymocytes using Qiagen RNeasy Mini Kit. cDNA was generated using Superscript IV Reverse Transcriptase (Invitrogen). cDNA was amplified and detected using TaqMan probes for Srebf1 (Thermo #Mm00550338_m1), Srebf2 (Thermo #Mm01306292_m1), Idi1 (Thermo #Mm01337454_m1), Sqle (Thermo #Mm00436772_m1), Nr1h2 (Thermo #Mm00437265_g1), Sult2b1 (Thermo #Mm00450550_m1), Ldlr (Thermo #Mm00440169_m1), Abcc1 (Thermo #Mm01344332_m1), Dhcr7 (Thermo #Mm00514571_m1), Hmgcr (Thermo #Mm01282499_m1), Abca1 (Thermo #Mm00442646_m1), Abcg1 (Thermo #Mm00437390_m1) as well as an 18S rRNA (Applied Biosystems #4352930) to serve as an internal control. A StepOnePlus Real-Time PCR system was used, and differences in abundance were calculated using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)).

Total RNA was isolated 96 hours after siRNA transfection and 72-96 hours after treatment with SUMO inhibitor using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed in quadruplicates to determine relative gene expression using TaqMan primer/probe combinations for TFAP2A, PIAS1 and CD44 with 18s rRNA for the endogenous control (Applied Biosystems).