alexa Whole Transcriptome Analysis with Ion Torrent Platform on Challenging Tissues: Formalin-Fixed, Paraffin-Embedded Tissues and Laser Capture Microdissected Samples
ISSN 2469-9853

Journal of Next Generation Sequencing & Applications
Open Access

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Research Article

Whole Transcriptome Analysis with Ion Torrent Platform on Challenging Tissues: Formalin-Fixed, Paraffin-Embedded Tissues and Laser Capture Microdissected Samples

Francesca Lessi1*, Paolo Aretini1, Sara Franceschi1, Marco La Ferla1, Samuele Negro2, Generoso Bevilacqua1 and Chiara Maria Mazzanti1

1Genomic Section, Pisa Science Foundation ONLUS, Via Panfilo Castaldi, 2, 56121, Pisa (PI), Italy

2Department of Biomedical Sciences, University of Padova, Italy

*Corresponding Author:
Francesca Lessi
Staff Scientist, Genomic Section
Pisa Science Foundation ONLUS
Via Panfilo Castaldi, 2, 56121, Pisa (PI), Italy
Tel: +39050974061
Fax: +390509656178
E-mail: [email protected]

Received date: June 09, 2016; Accepted date: October 14, 2016; Published date: October 16, 2016

Citation: Lessi F, Aretini P, Franceschi S, La Ferla M, Negro S, et al. (2016) Whole Transcriptome Analysis with Ion Torrent Platform on Challenging Tissues: Formalin-Fixed, Paraffin-Embedded Tissues and Laser Capture Microdissected Samples. Next Generat Sequenc & Applic 3:136. doi: 10.4172/2469-9853.1000136

Copyright: © 2016 Lessi F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Abstract

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.

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