Light cycler thermal cycler system

RT-qPCR was conducted in two steps. For the first-strand cDNA synthesis, 1 µg of total RNA was reverse-transcribed using random hexamers as primers and Transcriptor Reverse Transcriptase (QuantiTect^® Reverse Transcription kit (Qiagen)). Gene expression was assessed by real-time PCR using a LightCycler^® Thermal Cycler System (Roche Diagnostics). The reaction was carried out according to manufacturer’s protocol. The primers are listed in Table S1 (Supplementary Materials). The reactions consisted of an initial denaturing of 10 min at 95 °C, then 40 cycles of a 15 s denaturing phase at 95 °C, and a 1 min annealing and extension phase at 60 °C. The amplification curves were analyzed using the Roche LC software, both for determination of Ct and for melting curve analysis. Fidelity of the PCR was determined by melting temperature analysis. The gene expression values were calculated using the 2^−ΔΔCt method and normalized to the mean of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All measurements were performed in duplicate. Controls consisting of reaction mixture without cDNA were negative in all runs.

Analyses of renal klotho mRNA were performed by quantitative Real-Time PCR. Kidney tissue was disrupted using liquid nitrogen and grinded thoroughly with a mortar. Renal total RNA was extracted with using chloroform and isopropanol precipitation method and a treatment with DNAse I amplification Grade (Sigma-Aldrich) and quantified by spectrophotometry (ND-1000, Nanodrop Technologies, Wilmington, DE). Fifty ng of total RNA were used to analyze mRNA expression in the Light Cycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA). RT-PCR was performed in one step, with the QuantiTect SYBR Green RT-PCR kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. Rat primers for membrane Klotho were designed with the free Oligo 7 software (http://www.oligo.net/), and the sequence is F: 5´-CTCTGAAAGCCTACGTGTTGG-3´ and R: 5´-TAGAAACGAGATGAAGGCCAG-3´. Results were normalized to that of GAPDH. Primers sequence for GAPDH is F: 5´-AGGGCTGCCTTCTCTTGTGAC-3´ and R: 5´-TGGGTAGAATCATACTGGAACATGTAG-3´. Quantification of relative expression was determined by the 2(2ΔCt) method.

Total RNA was extracted by RNeasy Plus Mini Kit (Quiagen), according to manufacturer’s recommendations. RNA concentration was determined spectrophotometrically at 260 and 280 nm and RNA samples were stored at -80°C until use. One-step reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the QuantiTect SYBR Green RT-PCR kit (QIAGEN GmbH, Hilden, Germany) following manufacturer’s protocol. Expression levels of VEGF gene were measured by quantitative real-time RT-PCR using the LightCycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as housekeeping gene. Theoretical size of PCR products, sequence of primers used for study and efficiency were: VEGF: (254 bp), Forward primer: 5’-CCCTGATGAGATCGAGTACATCTT -3’; Reverse primer: 5’-AGCAAGGCCCACAGGGATTT-3, Efficiency: 2; GAPDH: (240 bp), Forward primer: 5’-TGATGACATCAAGAAGGTGGTGAAG-3’;
Reverse primer: 5’-TCCTTGGAGGCCATGTAGGCCAT-3’, Efficiency: 2; Results were normalized to that of GAPDH and quantification of relative expression was determined by the 2^-ΔΔCt method.

Total RNA was isolated using the TRIzol reagent protocol (Invitrogen, Thermo Fisher Scientific, Walthan, MA, USA). The sequence of primers used for RT-PCR is shown in Table S1. Quantification was done using the QuantiTect SYBR Green RT-PCR kit (Qiagen GmbH, Hilden, Germany) for 50 ng of RNA and 1 µL of primer. The mRNA expression was analyzed in the Light Cycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA) and the relative expression of the target genes was determined using the 2^−ΔΔCt method.

A human atherosclerosis RT² Profiler PCR array (Qiagen, Hilden, Germany) was used to profile the expression of 84 genes related to atherosclerosis. The description of this PCR array can be found on the following website: http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-038Z.html (1 January 2016). Specifically, single-stranded cDNA was synthesized from 500 ng of total RNA from two samples (each consisted of a pool of three RNA samples of PBMCs from r-axSpA patients or healthy donors (HDs)), using an RT² first-strand cDNA synthesis kit (Qiagen). Next, cDNAs were mixed with RT² SYBR Green qPCR Mastermix (Qiagen), and real-time PCR was performed according to the manufacturer’s protocol. Quantitative real-time PCR was performed on a LightCycler^® Thermal Cycler System (Roche Diagnostics, Indianapolis, IN, USA). Four housekeeping genes (i.e., β-actin, b2-microglobulin, hypoxanthine phosphoribosyl transferase 1, and ribosomal protein large p1) were used for normalization. The gene expression values were calculated using the 2^−ΔΔCt method. All measurements were performed in duplicate.

Changes of DDX11 were validated in an independent cohort of 31 SLE patients and 32 healthy donors (HD) from Cordoba, Spain (Table 1) by quantitative real-time RT-PCR using a LightCycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA), using GAPDH as housekeeping gene. Specific primers for DDX11 were previously reported [23 (link)]. These samples were collected after obtaining approval from the ethics committee of the Reina Sofia Hospital, Cordoba. All subjects provided written informed consent. Briefly, RT was performed using an NZY First-Strand cDNA Synthesis Kit Reverse Transcription Kit (Nzytech, Lisbon, Portugal), following the manufacturer’s instructions. For the qPCR, the LightCycler thermocycler system (Roche Diagnostics, Indianapolis, IN, USA) was used. The reaction was carried out with SYBR^® Green (Promega Biotech, Madrid, Spain) according to the manufacturer’s instructions. Expression of DDX11 was corrected by the geometric average of α-actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data were analyzed by the 2-ΔΔCt method. The expression of DNM1L and KRAS was similarly assessed in 20 SLE cases and 20 HD controls, as described for DDX11 above.