Automated TruSeq RNA Sample Preparation from FFPE tissue specimens utilizing the Biomek FXp Liquid Handler

Next Generation Sequencing (NGS) technology facilitates high throughput, high speed and cost-effective sequencing of DNA and/or RNA. One key application of NGS is to analyze whole transcriptome of cells. Analysis of transcriptome reveals the gene expression information, detects post transcriptional mutations, quantification of gene expression profiling in a certain disease development pathway. This application note describes automated RNA library construction using the Beckman Coulter Biomek FXP Liquid Handler from archival FFPE tissue specimens. The RNA is extracted using the Beckman Coulter Agencourt FormaPure kit and the library preparation method is based on the Illumina TruSeq RNA sample preparation protocol (P/N 15026495 Rev C). The Biomek TruSeq RNA method comprises three parts; mRNA purification, fragmentation and cDNA synthesis, cDNA library construction (end repair, A-tailing and adaptor ligation), and PCR amplification and product purification. This automated method is suitable for up to ninety-six library constructions. The data presented here benchmarks the novel automated approach using a Biomek FXP Liquid Handler against a standard manual preparation. The archival FFPE tissue specimens were pathologically reviewed and assigned as tumor or benign regions of interest RNA isolated from archival FFPE tissue is highly degraded due to a combination of fixation conditions, temperature, humidity and age. Therefore the RNA input material for a library construction from FFPE tissue is typically of lower yield and poorer quality than fresh frozen samples. The findings of our method development study were that libraries prepped on the Biomek automated platform were significantly superior to their manually prepped counterparts and that ultimately this provides end-users with a more reliable way to generate the libraries from precious clinical samples in a faster turnaround time.