Rna ffpe tissue extraction kit

Tumors underwent RNA sequencing using full formalin-fixed paraffin-embedded (FFPE) specimens that were reviewed by a board-certified pathologist to measure percent tumor content and tumor size; a minimum of 20% of tumor content in the area for microdissection was required to enable enrichment and extraction of tumor-specific RNA. A Qiagen RNA FFPE tissue extraction kit was used for extraction, and the RNA quality and quantity were determined using the Agilent TapeStation. Biotinylated RNA baits were hybridized to synthesized and purified cDNA targets and the bait-target complexes were amplified in a post-capture PCR reaction. The Illumina NovaSeq 6500 was used to sequence the whole transcriptome from patients to an average of 60 M reads. Raw data was demultiplexed by Illumina Dragen Bio-IT accelerator, trimmed, counted, PCR-duplicates removed, and aligned to the human reference genome hg19 by STAR aligner. For transcription counting, transcripts per million molecules were generated using the Salmon expression pipeline. Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA) was prepared. Immune cell fraction was calculated by QuantiSeq using this transcriptomic data^{48 (link)}. Additionally, this mRNA data was used as input for pathway gene enrichment analyses using Gene Set Enrichment Analysis^{49 (link)}.

Gene fusion detection was performed on mRNA isolated from a FFPE tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, Santa Clara, CA). FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required to enable enrichment and extraction of tumor-specific RNA. Qiagen RNA FFPE tissue extraction kit was used for extraction, and the RNA quality and quantity was determined using the Agilent TapeStation. Biotinylated RNA baits were hybridized to the synthesized and purified cDNA targets and the bait-target complexes were amplified in a post capture PCR reaction. The resultant libraries were quantified, normalized and the pooled libraries are denatured, diluted and sequenced; the reference genome used was GRCh37/hg19 and analytical validation of this test demonstrated ≥97% Positive Percent Agreement (PPA), ≥99% Negative Percent Agreement (NPA) and ≥99% Overall Percent Agreement (OPA) with a validated comparator method.