Francesca Lessi, Paolo Aretini, Sara Franceschi, Marco La Ferla, Samuele Negro, Generoso Bevilacqua and Chiara Maria Mazzanti
Background: The advent of the NGS revolutionized cancer research by making it possible to study the complexity of cancer using high throughput sequencing methodologies. The current trends are to adapt highthroughput sequencing technologies to the level of small cell populations and even individual cells. In our laboratory, we developed some different methods that would allow us to work optimally with very low amount of material, based on the technique of the SMARTer technology and on the Ion Torrent protocols that we modified carefully, applied to Ion Proton system.
Materials and methods: We collected 12 formalin-fixed, paraffin-embedded tissues and 20 laser capture microdissected formalin-fixed, paraffin-embedded samples derived from brain and breast cancer and 13 laser capture microdissected fresh frozen samples derived from mouse brain cells.
Results: We developed high performance methods to analyse the whole transcriptomes of our samples, obtaining a very high number of reads (78,186,377 usable reads), perfectly comparable with samples with a large amount of RNA such as samples obtained from cells or fresh tissues.
Conclusions: We have found that the combination of SMARTer technology and Ion TargetSeq Exome Enrichment kit, in addition to some improvements to their conventional protocols, provides excellent results with challenging samples.