RNAseq-differentiated gene expression profile of people living with HTLV-1 in the Brazilian Amazon region: a pilot study

To investigate the differential expression profile of genes and microRNA (miRNA) in peripheral blood mononuclear cells (PBMCs) from patients infected with Human T-lymphotropic Virus 1 (HTLV-1), as well as their impact on progression to Tropical Spastic Paraparesis /HTLV Associated Myelopathy (PET/HAM) Overall design: Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll from 4 individuals with HAM, 3 asymptomatic individuals and 3 HTLV seronegative blood donors (control group). After RNA extraction by phycol-chloroform-isopropanol, quantification of the key proteins of the Dicer and Drosha miRNA pathway was performed by RTqPCR (SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR). The ß-actin cellular normalizing gene was used as an endogenous control to ensure RNA integrity of the extracted samples. The mRNA quantifications were performed by the 2-??CT method. To obtain the transcriptome, the samples were quantified (Qubit™ RNA BR Assay Kit) and their integrity evaluated with (Agilent RNA 6000 Pico Kit). Samples that had an RIN (RNA Integrity Number) greater than 7 were selected and processed for making the cDNA library according to the SureSelect Strand-Specific RNA Library Prep System guide from the Illumina platform. Sequencing was performed on the Illumina NextSeq 500 equipment. Differentially expressed genes were obtained using the EdgeR package, based on the False Discovery Rate (FDR= 0.05).