GSE130512 ( miRNA )

tyriod cancer ( tyriod )

Diagnostic value of a panel of plasma exosomal miRNAs as potential biomarkers for thyroid nodules

Cancer : 16 / Control : 8

The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia). After cluster generation, the libraries were sequenced and 50bp single-end reads were generated. The raw data (raw reads) extracted from FASTQ files were firstly processed through perl and python scripts. In this step, clean data(clean reads) were generated by removing reads containing ploy-N, with 5’ adapter contaminants, without 3’ adapter or the insert tag, containing ploy A or T or G or C and low quality reads from raw data.Then the clean data were aligned against human miRNA sequences from miRBase (Release 20) and NCBI human genome reference sequences (Release 103) using Bowtie (version 0.12.8) . miRNA profiling was normalized using read counts of a specific miRNA per million(TPM) mappable miRNA sequences. To find novel miRNAs, the raw data was independently processed by combined use of miRDeep 2 and miREvo. Predicted miRNAs with scores ≥ 2 were considered significant.

Exosomes and exosomal miRNAs from the plasma of volunteers were isolated from thyroid nodules and papillary tyriod cancer patients . Profiling of exosomal miRNA was performed using next-generation sequencing(NGS) to identify miRNA candidates for diagnosis.

Sequencing small RNA libraries prepared from 8 benign thyroid nodules and 16 PTC patients with or without metastasis.

Liang M, Yu S, Tang S, Bai L et al. A Panel of Plasma Exosomal miRNAs as Potential Biomarkers for Differential Diagnosis of Thyroid Nodules. Front Genet 2020;11:449. PMID: 32508877

Small RNA libraries were constructed as instructed by the NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, Ipswich, MA, USA) with minor modifications. Ten ng of small RNA were used as the starting material. After sequential ligation of the Multiplex 3’ SR Adaptor, hybridization of the reverse transcription primer, and ligation of the Multiplex 5’ SR Adaptor, the RNA-Adaptor complex was reversed transcribed into cDNA. Then the small RNA libraries were amplified and the Illumina compatible Index Primers were incorporated with 10 cycles of PCR amplification. The amplified libraries were resolved on a native 5% acrylamide gel (Bio-rad, Hercules, CA). DNA fragments corresponding to 140 - 160 bp (the size comprising the miRNA inserts plus the 3’ and 5’ adaptors) were recovered in 12 µL of Elution Buffer (QIAGEN) and determined with High Sensitivity DNA Chip on Agilent 2100 bioanalyzer. Absolute quantities of each indexed library were determined by real time qPCR using KAPA Library Quantification Kit according to the manufacture’s protocol.

total RNA

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