GSE122488 ( miRNA )

glioblastoma ( glioma )

Deep sequencing of circulating exosomal microRNA allows non-invasive glioblastoma diagnosis

Cancer : 22 / Control : 16

Illumina Casava1.7 software used for basecalling. Data pre-processing was performed using a pipeline comprising of adapter trimming (cutadapt), followed by genome alignment to human genome hg 19 using Bowtie (18 bp seed, 1 error in seed, quality score sum of mismatches<70). Where multiple best strata alignments existed, tags were randomly assigned to one of those coordinates. Tags were annotated against mirBase 20 and filtered for at most one base error within the tag. Counts for each miRNA were tabulated and adjusted to counts per million miRNAs passing the mismatch filter.Supplementary_files_format_and_content: All samples achieved miRNA read counts >45,000 read counts and miRNAs with low abundance (<50 read counts across more than 20% of samples) were removed.

Serum exosomal-microRNA isolated from IDH-wildtype glioblastoma (n=12) and IDH-mutant glioma grades II-III (n=10) were analyzed using small-RNA next generation sequencing and compared to age- and gender-matched healthy controls. Differentially expressed miRNAs (|fold change|³2 and p-value ≤ 0.05 in three statistical tests, Fischer-exact, t-test, and Wilcoxon) were identified and the predictive power of individual and subsets of miRNAs were tested using univariate (logistic regression) and multivariate (Random Forest) analyses. Additional glioblastoma sera (n=4) and independent sets of healthy (n=9) and non-glioma (n=10) controls were used to further test the predictive power of our glioblastoma miRNA signature.

Serum exosomal miRNA profiles ofIDH-wildtype glioblastoma, IDH-mutant glioma grades II-III, their matched healthy controls, and additional GBM patients, non_GBM patients and healthy controls were generated by deep sequencing, single end, using Illumina HiSeq. 2000 System at the Ramaciotti Centre for Genomics.

Ebrahimkhani S, Vafaee F, Hallal S, Wei H et al. Deep sequencing of circulating exosomal microRNA allows non-invasive glioblastoma diagnosis. NPJ Precis Oncol 2018;2:28. PMID: 30564636 // Hallal S, Ebrahim Khani S, Wei H, Lee MYT et al. Deep Sequencing of Small RNAs from Neurosurgical Extracellular Vesicles Substantiates miR-486-3p as a Circulating Biomarker that Distinguishes Glioblastoma from Lower-Grade Astrocytoma Patients. Int J Mol Sci 2020 Jul 13;21(14). PMID: 32668808

Exosomes were isolated from 1 mL RNase A (37 °C for 10 min; 100 ng/ml; Qiagen, Australia) treated serum, by size exclusion chromatography (qEV iZONE Science). Then, Serum exosomes were processed for RNA extraction using the Plasma/Serum Circulating & Exosomal RNA Purification Mini Kit (Norgen Biotek, Cat. 51000) according to the manufacturer’s protocol. Exosome RNA sequencing libraries were then constructed using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina (BioLabs, New England) according to the manufacturer’s instructions.

total RNA

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