Dna free kit

For microarray analysis and Northern blotting total RNA was extracted from the cell pellets using IsolRNA (5prime) and DNA was removed using the DNA-free kit (Applied Biosystems). DNase-treated total RNA was subsequently run through RNeasy MinElute Cleanup columns (Qiagen) to remove any remaining contaminants, and to further concentrate the RNA. The RNA was finally dissolved in 16 μl RNase free water.

Following tissue harvesting, half of the placentas were preserved in RNAlater at −20°C. Tissue homogenization was carried out using micro packaging vials with ceramic beads (1.4 mm) in a Precellys® 24 Tissue Homogenizer (Peqlab). RNA isolation and DNA digestion were conducted by use of RNeasy Plus Universal Mini Kit (QIAGEN) and DNA-free Kit (Applied Biosystems by Thermo Fisher), respectively. cDNA synthesis was performed with random primers (Invitrogen by Thermo Fisher). Concentration and purity of RNA and cDNA were assessed employing NanoQuant (Tecan).

We performed RNA isolation and cDNA production similarly to what has been previously reported by our lab [17 (link)]. Briefly, we homogenized frozen tissue in 1 ml of Tri-reagent (Molecular Research Center, P/N TR 118). RNA was then isolated following the manufacturer’s directions and quantified for concentration using a Nanodrop 2000. The mean concentration obtained was 319.3 ng/μl. The mean 260/280 ratio was 1.89 and the mean 260/230 ratio was 1.52. The integrity of the RNA was assessed using an Agilent 2100 BioAnalyzer (Agilent Technologies) using the manufacturer’s instructions, with a mean integrity of 7.94 ± 0.01 (scale 1–10, 10 being perfect) and the mean 28S-to-18S ratio at 1.35 ± 0.004. The RNA was then treated with deoxyribonuclease (DNase) (Applied Biosystems DNA-free kit, #AM1906), following the manufacturer’s instructions, to remove any endogenous genetic DNA. Then 3 μg of purified RNA underwent the reverse transcriptase reaction to produce cDNA (Bio-Rad iScript kit, #170-8891), following the manufacturer’s instructions, scaled to a reaction volume of 60 μl, using a BioRad iQ5 to run the reaction (protocol: 25 °C for 5 min; 42 °C for 30 min and 85 °C for 5 min). The cDNA was stored at −80 °C.

Using the RNeasy-Mini Kit (Qiagen Inc., Valencia, CA, USA), total RNA from bladder tissues was isolated, and the DNA-free Kit (Applied Biosystems, Foster City, CA, USA) was used to remove genomic DNA. Using Taqman reverse transcription reagents (Applied Biosystems), mRNA (400 ng) was reverse transcribed per manufacturer’s instructions. Quantitative assessment of the target genes’ expression levels was performed using real-time quantitative PCR (RQ-PCR) with a PikoReal™ Real-Time PCR System (Thermo Scientific) with iQ™ SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA), as described previously^{39 (link),40 (link)}. Gene expression data were quantified using duplicated RQ-PCR assays (n = 10) from five independent animals.

Total RNAs from blood leukocytes and human umbilical vein endothelial cells (HUVEC) were extracted with TRIzol (Invitrogen, Saint-Aubin, France) and a commercial kit (Macherey Nagel, Hoerdt, France), respectively. Genomic DNA was removed using DNA-free kit (Applied Biosystems, Life Technologies, Saint Aubin, France) and first-strand reverse transcription (Applied Biosystems) was performed. Real-Time PCR assays were performed on a RotorGene Q (Qiagen, Courtaboeuf, France) following the manufacturer’s recommendations. Human DNA primers were designed using OligoPerfect™ (Invitrogen), QuantPrim (Universität Potsdam, Max-Planck-Gesellschaft) and OligoAnalyser (Integrated DNA Technologies, Inc) with the sequences detailed in Table S1. mRNA expression levels in the samples relative to expression in day 0 were determined with the Pfaffl method (expressed as Relative Fold Change), using ribosomal L19, S9 and RPLPO genes as internal controls. mRNA sST2 levels were assessed using sST2/ST2L ratio (sST2 primer detected a common region of soluble and membrane ST2 while ST2L primer detected only the membrane region).