Abstract 5781: Development of RNA-Seq library preparation method for highly degraded FFPE RNA and ultra-low RNA input

Abstract RNA sequencing (RNA-Seq) has been widely used for investigating genetic variations, splicing, and gene expression in cancer research. However, RNA-Seq library preparation from archived formalin fixed paraffin embedded (FFPE) samples tends to fail due to both limited quantity and high degradation. We aimed to overcome the sample quality and quantity limitations by leveraging both in-house cDNA synthesis modules and the unique, single-stranded ligation strategy of the IDT xGen cfDNA 3) from lung, breast, colorectal, thyroid tumors and more. In addition, twenty-one frozen tissue RNA samples and Nine RNA reference materials were used as controls. Regardless of sample origin, 25 ng of total RNA were taken for RNA-Seq library preparation. In-house cDNA synthesis modules were used for the 1st and 2nd strand cDNA synthesis followed by Illumina library generation using IDT’s xGen™ cfDNA 1 ug) for hybridization capture enrichment. The RNA-seq libraries were captured using IDT’s custom designed panel with 56 known fusion targets. We detected all expected fusion targets from the tested samples. No correlation was observed between sample quality and the number of genes detected between the different types of samples including the low quality FFPE. To test the lower limits of the workflow, a degraded FFPE sample and a Universal Human Reference RNA (UHR) were used to construct libraries with inputs from 1ng to 10pg. High quality libraries were successfully constructed with as low as 10pg of UHR and 50pg of the FFPE RNA sample. We demonstrated the flexibility of our current cfDNA Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5781.