Tissue was cut into 5µm serial sections and mounted on glass microscope slides. Diagnosis was confirmed by a board-certified pathologist specialized in thyroid disease and regions of interest representing tumor foci on an H&E slide were selected and circled. Samples were sent to HTG Molecular Diagnostics in Tucson, AZ, and regions of interest were microdissected under a Leica LMD6500 laser capture microscope (LCM). Each sample was immediately suspended in EdgeSeq lysis buffer (HTG Molecular Diagnostics, Tucson, AZ) and miRNA/mRNA profiling was carried out using one FFPE tissue slide from each sample for each assay as previously described (25 (link)). miRNA and mRNA expression were profiled using HTG Edgeseq miRNA whole transcriptome assay and HTG EdgeSeq Oncology Biomarker Panel (OBP), respectively. HTG EdgeSeq system is a NGS application that measures gene expression without the need for extracting RNA. HTG Edgeseq miRNA whole transcriptome assay measures the expression of 2,083 human miRNAs described in the miRBase v20 database. HTG EdgeSeq OBP measures the expression of 2,559 genes associated with tumor biology, including 15 housekeeping genes. All miRNAs and mRNAs screened in this study are listed in Supplemental Tables 1, 2, respectively. Briefly, the workflow entailed automated, extraction-free sample preparation, quantitative nuclease protection using the EdgeSeq processor, and library generation and sequencing. The HTG EdgeSeq Parser (HTG Molecular, Tucson, AZ, USA) was used to align the FASTQ files to the probe list to collate the data. Data were provided as data tables of raw, quality control (QC) raw, counts per million (CPM), and median normalized counts.
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