Oligonucleotide primers

Total RNA was extracted from the renal tissue of the SHAM or IRI-mice that were treated or not with HLSC-EVs using TRIzol™ reagent (Ambion, Thermofisher, Waltham, MA, USA), following the manufacturer’s instructions. A Bullet Blender instrument (Next Advance Inc., New York, NY, USA) was used to homogenize TRIzol™ solutions using 0.5 mm zirconium oxide beads at a speed of 8 rpm for 3 min, followed by centrifuge at 12,000× g for 10 min at 4 °C. RNA that was purified from the supernatant of the homogenized tissue was quantified spectrophotometrically (mySPEC, VWR, Radnor, PA, USA).
A High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to convert RNA into cDNA. A 96-well QuantStudio 12K Flex Real-Time PCR system (Thermo Fisher Scientific) was used to evaluate specific gene expression by quantitative Real-Time PCR (qRT-PCR). Each well was loaded with 20 µL reaction mixture containing Power SYBR Green PCR Master Mix (Applied Biosystems), 5 or 10 ng of sample cDNA, and sequence-specific oligonucleotide primers (100 nM, purchased from MWG-Biotech, Eurofins Scientific, Brussels, Belgium). All the primers that were used for qRT-PCR are listed in Table 1. To normalize the RNA inputs, GAPDH was used as housekeeping gene. For all samples, fold-change expression with respect to the IRI group or the SHAM group was calculated using ΔΔCt method.

Acute dengue infection was confirmed by quantitative real time PCR and DENV viruses were serotyped and titres quantified by quantitative real time PCR as previously described [18 (link)]. Multiplex quantitative real-time PCR was performed as using the CDC real time PCR assay for detection of the dengue viruses [19 (link)], and modified to quantify DENV. Oligonucleotide primers and a dual labeled probe for DENV 1,2,3,4 serotypes were used (Life technologies, India) based on published sequences [19 (link)].