Comparison of the Effects of Tumor Preservation Techniques on Single Nuclei Isolation
Intra-tumor heterogeneity (ITH), genetic and transcriptomic, is one of the hallmarks of tumor “ecosystems”. Increased ITH is associated with worse prognosis and increased drug resistance in cancer patients. Current next-generation sequencing technologies have revealed extensive heterogeneity at the molecular level in many cancers. Due to the complex cellular profiles and issues that arise because of ITH, it is essential to analyze tumors at the single cell level to provide appropriate and efficient personalized treatments against these cancers. However, the enzymatic dissociation processes used to isolate single cells has been shown to influence the transcriptomic profile of the cells involved. Hence, single nuclei isolation has evolved as a preferred method for transcriptomic analysis, as it circumvents enzymatic dissociation for nuclear isolation, and therefore maintains transcriptome integrity.
Single nuclei sequencing has gained immense popularity in various transcriptomic studies, especially when considering highly interconnected tissues, such as glioblastoma multiforme (GBM). However, extracting quality nuclei and RNA for sequencing from preserved tissue such as formalin fixed and paraffin embedded (FFPE) and flash frozen (FF) remains a challenge. The purpose of this project is to compare the effects of varying preservation techniques on the transcriptome of tumor cells. Here, we demonstrate optimized nuclei isolation techniques used to obtain intact nuclei with minimal debris while simultaneously maximizing the yield and quality of RNA.