Effects of Duplex-specific nuclease on human cells expression profiling using RNA-seqV. Mijatovic 1 , G. Buson 2 , M. Valenti 3 , L. Dalle Carbonare 3 , A. Ferrarini 2 , A. Dal Molin 1 , A. Mori 1 , L. Xumerle 1 , E. Caviola 4 , P. Pertile 4 , M. Delledonne 2 , P. F. Pignatti 1 , G. Malerba 1 ;1 Department of Life and Reproductions Sciences, University of Verona, Verona, Italy, 2 Department of Biotechnology, University of Verona, Verona, Italy, 3 Department of Medicine, Clinic of Internal Medicine, Section D, Verona, Italy, 4 Cutech Srl, Padova, Italy.RNA-seq is a next-generation sequencing method able to characterize tho-ence the estimate of relative expression of less expressed genes, as well as reduce the estimate accuracy of gene expression levels of low expressed genes. Therefore, it would be convenient to apply a method able to reduce excessively abundant mRNA transcripts. A cDNA depletion method for the most represented transcripts called duplex-specific nuclease (DSN) has been effectively used in several studies. Three different cell types were studied: blood, monocytes and keratinocytes. From each cell type two RNA-seq libraries were produced (untreated libraries), for a total of 6 libraries. A part of each library was then treated with DSN, producing a total of 6 DSN treated libraries. Overall, 12 RNA-seq libraries (6 untreated and 6 treated) were prepared. In blood tissue the most abundant transcripts in both DSN-treated and DSN-untreated samples corresponded to globins (HBA2, HBA1 and HBB), accounting for ~70% of total transcripts. In this sample maximum effects of DSN treatment was observed. Transcripts found to be the most expressed in monocyte sample were ~10 times smaller than globins expression in blood sample. Gene expression of keratinocyte libraries was not influenced by DSN treatment. DSN treatment has been able to strongly reduce globins expression. DSN treatment also reduced, even if with a smaller effect, the most expressed genes in monocyte sample.

Effects of Duplex-specific nuclease on human cells expression profiling using RNA-seq

V. Mijatovic;G. Buson;M. T. Valenti;L. Dalle Carbonare;A. Dal Molin;A. Mori;L. Xumerle;M. Delledonne;P. F. Pignatti;G. Malerba
2014

Abstract

Effects of Duplex-specific nuclease on human cells expression profiling using RNA-seqV. Mijatovic 1 , G. Buson 2 , M. Valenti 3 , L. Dalle Carbonare 3 , A. Ferrarini 2 , A. Dal Molin 1 , A. Mori 1 , L. Xumerle 1 , E. Caviola 4 , P. Pertile 4 , M. Delledonne 2 , P. F. Pignatti 1 , G. Malerba 1 ;1 Department of Life and Reproductions Sciences, University of Verona, Verona, Italy, 2 Department of Biotechnology, University of Verona, Verona, Italy, 3 Department of Medicine, Clinic of Internal Medicine, Section D, Verona, Italy, 4 Cutech Srl, Padova, Italy.RNA-seq is a next-generation sequencing method able to characterize tho-ence the estimate of relative expression of less expressed genes, as well as reduce the estimate accuracy of gene expression levels of low expressed genes. Therefore, it would be convenient to apply a method able to reduce excessively abundant mRNA transcripts. A cDNA depletion method for the most represented transcripts called duplex-specific nuclease (DSN) has been effectively used in several studies. Three different cell types were studied: blood, monocytes and keratinocytes. From each cell type two RNA-seq libraries were produced (untreated libraries), for a total of 6 libraries. A part of each library was then treated with DSN, producing a total of 6 DSN treated libraries. Overall, 12 RNA-seq libraries (6 untreated and 6 treated) were prepared. In blood tissue the most abundant transcripts in both DSN-treated and DSN-untreated samples corresponded to globins (HBA2, HBA1 and HBB), accounting for ~70% of total transcripts. In this sample maximum effects of DSN treatment was observed. Transcripts found to be the most expressed in monocyte sample were ~10 times smaller than globins expression in blood sample. Gene expression of keratinocyte libraries was not influenced by DSN treatment. DSN treatment has been able to strongly reduce globins expression. DSN treatment also reduced, even if with a smaller effect, the most expressed genes in monocyte sample.
Next generation sequencing; RNA-seq; Duplex-specific nuclease
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/921194
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