title: New Insights into the Complex Pathophysiology of Restless Legs Syndrome from a Transcriptome Analysis of mRNAs
design: New Insights into the Complex Pathophysiology of Restless Legs Syndrome from a Transcriptome Analysis of mRNAs
library_construction_protocol: For this study 17 drug-free patients (13 F and 4 M, mean age 55.8 years, range 24-76; mean IRLS severity scale 21.1; mean disease duration 6.6 years) were enrolled, along with 18 age- and sex-matched controls. RLS was diagnosed according to the International RLS Study Group diagnostic criteria (Allen et al. 2014) by means of a semi-structured clinical interview and a careful exclusion of RLS mimics. RNA extraction was performed as described in Salemi et al.  The RNA was stored at -80° C until further processing.( PMID: 35163455) RNA sequencing and data analysis were performed by Genomix4Life Srl (Baronissi, Italy). The yield and quality of RNA were evaluated as described above in Salemi et al. (32). Indexed libraries were prepared from 600ng/ea purified RNA with Illumina Stranded mRNA (Illumina) Library Prep Kit according to the manufacturer's instructions.  After enrichment of mRNA by oligo dT magnetic beads, purified mRNA is fragmented and copied into first strand complimentary DNA (cDNA) using reverse transcriptase and random primers. In a second strand cDNA synthesis step, dUTP replaces dTTP to achieve strand specificity, followed by adapter ligation and PCR amplification