Simultaneous Interrogation of Small RNAs and Long RNAs from archival breast cancer FFPE samples using Next Generation Sequencing (NGS)

Simultaneous Interrogation of Small RNAs and Long RNAs from archival breast cancer FFPE samples using Next Generation Sequencing (NGS)

Abstract
RNA expression studies in oncology are critical for diagnosis, therapy decisions and prognosis. Availability of fresh tumor specimens for high quality analysis is rare, especially, for long-term outcome studies. This limitation is overcome by treating the collected tumor tissues with formaldehyde solution and embedding them in paraffin wax. FFPE blocks can be stored for long periods of time at room temperature. The process of retrieving RNA from FFPE sections damages the RNA impacting the quality of gene detection. Less than twenty percent of the fragments from FFPE samples are greater than 200bp. Traditional ligation-based library construction method is not equipped to handle smaller fragments and impacts the final library quality. Thus, a robust library preparation workflow with a novel enzyme chemistry that can make-up for the sample limitation is key to unlocking the potential of FFPE samples for RNA-Seq.
In this presentation, we report an effective RNA-Seq method to interrogate small RNAs and long RNAs from archived breast cancer FFPE tissues. We demonstrate the efficiency of using a novel library preparation method using engineered retrotransposon enzyme called SEQzyme. This enzyme’s unique modus operandi combines cDNA synthesis and adapter ligation in one continuous synthesis reaction and drastically reducing workflow times.
In our study, we compared two different extraction methods for FFPE samples. Our FFPE data was benchmarked against matching fresh frozen samples. The total number of genes detected was similar across the workflows and between FFPE and fresh frozen samples. We utilized a custom data analysis pipeline designed for use with our workflow enabling detection of RNAs greater than 20 bp. We showcase detection of different RNA biotypes – mRNAs, lncRNAs, and smRNAs. Significantly greater number of miRNAs were captured with our method.
We demonstrate enhanced recovery of smaller fragments from degraded FFPE sample and enable a more complete transcriptome analysis. By unlocking more information from FFPE samples using RNA-Seq we demonstrate the gene expression in breast cancer samples. This study can be extended to other sample types to impact translational disease research.