hum0069-v1Release info

RNA expression profiling of neuromuscular diseases and viral diseases

Aims

MicroRNAs (miRNAs), particularly those found in human body fluids, have been suggested as potential biomarkers. Among various body fluids, the cerebrospinal fluid (CSF) shows promise as a profiling target for diagnosis and monitoring of neurological diseases. However, relevant genome-scale studies are limited and no studies have profiled exosomal miRNAs in CSF. Therefore, we conducted a next-generation sequencing-based survey of small RNAs in the exosomal and non-exosomal (supernatant) fractions of healthy human CSF as well as serum in each donor. Our data provided the first landscape of small RNAs in CSF exosome.

Methods

Samples were derived from three donors and subjected to a NGS-based survey. CSF samples were obtained via lumbar puncture, centrifuged to remove contaminating cells. Corresponding serum samples were simultaneously isolated from peripheral blood. Samples were further divided into exosomal and supernatant fractions via ultracentrifugation. Total RNA was isolated from each individual fractionated sample (Qiagen). Small RNA libraries were prepared using a TruSeq Small RNA Library Prep Kit (Illumina). Individual libraries were prepared with unique indexes, pooled and subjected to 50-base reads in single lanes of a HiSeq 2500 system (Illumina). Small RNA sequences were aligned to the human reference genome (hg19) using BWA (version 0.5.9). Small RNA clusters were annotated as miRNAs when their genomic coordinates overlapped with those of miRNAs registered in miRbase (version 20). The number of reads aligned within each individual cluster was normalized to counts per million (CPM) after applying a normalization factor based on the relative log expression method via edgeR. Post-processed reads were also analysed using the script 'quantifier.pl' in the miRdeep2 package (version 2.0.0.7; based on the miRbase database).

Participants/materials

CSF and serum samples from three donors (neuromuscular disease patients) were analyzed.

URL

• https://www.tmd.ac.jp/med/nuro/research-e.html (opens in a new tab)

Principal investigator

Takanori Yokota

Affiliation

Tokyo Medical and Dental University, Department of Neurology and Neurological Science