Illumina HiSeq 2500 paired end sequencing; Decreased miRNA expression in Klinefelter syndrome
Decreased miRNA expression in Klinefelter syndrome
This study was approved by the Ethics Committee of University teaching Hospital of “Policlinico-Vittorio Emanuele, University of Catania, (Catania, Italy), trial registration number 49/2015/PO (Register of the Ethics Committee opinions). All methods were performed in accordance with the relevant guidelines and regulations. All participants were asked for and provided their informed consent. We performed a transcriptome analysis on PBMCs of 10 non-mosaic KS patients and 10 aged-matched healthy controls. All KS patients (mean age 32.5±3.0 years) had 47,XXY karyotype as shown by cytogenetic investigation conducted on at least 50 metaphase and five out of ten were treated with testosterone enanthate. Normozoospermic controls (mean age 32.0±3.0 years) had a negative history for genetic disease, normal testicular volume and normal reproductive hormones (FSH, LH, total testosterone) levels. Ten healthy age-matched women (mean age 32.0±2.9 years) were also recruited as female controls.  All KS and male and female controls were Italians (Eastern Sicily) Heparinized venous blood sample was withdrawn from each KS and male and female control. Peripheral blood mononuclear cells (PBMCs) were obteined using Ficoll-Paque (Ficoll Plaque PLUS – GE Healtcare Life Sciences, Piscataway, USA). Total RNA was extracted using the standard RNA extraction method with TRIzol (Invitrogen, Carlsbad, CA, USA). Before use, RNA concentration in each sample was assayed with a ND-1000 spectrophotometer (NanoDrop) and its quality assessed with the Agilent 2100 Bioanalyzer with Agilent RNA 6000 nano kit (Agilent Technologies, Santa Clara, CA, USA).  Indexed libraries were prepared from 1 µg/ea purified RNA with TruSeq Stranded Total RNA (Illumina) Library Prep Kit according to the manufacturer's instructions. Libraries were quantified using the Agilent 2100 Bioanalyzer (Agilent Technologies) and pooled such that each index-tagged sample was present in equimolar amounts, with final concentration of the pooled samples of 2bnM