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Abstracts / Human Immunology 74 (2013) 51–173
IDENTIFICATION BY RNA-SEQ OF NOVEL ANCESTRAL MEDIATOR TRANSCRIPTS IN ENDOTHELIAL PROGENITORS. Monica Rienzo 1, Antonietta Picascia 2, Concetta Schiano 3, Amelia Casamassimi 1, Vincenzo Grimaldi 2, Claudio Napoli 1,2. 1 Department of General Pathology, Division of Clinical Pathology and Excellence Research Centre on Cardiovascular Disease, Second University of Naples, Naples, Italy; 2 U.O.C. Division of Immunohematology, Transfusion Medicine and Transplant Immunology (SIMT), Regional Reference Laboratory of Transplant Immunology (LIT), Second University of Naples, Naples, Italy; 3 Institute of Diagnostic and Nuclear Development, SDN Foundation, Naples, Italy. Aim: Mediator (MED) complex functions as a pivotal adaptor between transcription factors bounded at gene regulatory elements, RNA polymerase II and general transcription factors. Different ancestral humanMED complexes including at least 30 distinct MED subunits (MEDs) have been isolated. Because of the importance of ancestral MED role in the transcription of the eukaryotic genes, disruption of MED function may have relevant pathophysiological consequence also in the cardiovascular system and transplantation. Here, we have analyzed the expression data relative to MEDs in human endothelial progenitor cells (EPCs) obtained by RNA-Seq on a next generation sequencing platform. The introduction of next generation sequencing (NGS) technologies has revealed the complexity of mammalian transcriptomes, enabling to effectively explore, with an unprecedented throughput capacity, simple and complex genomes and even their differences in health and disease conditions. Methods: RNA was isolated from early human EPCs and after ribodepletion it was used for the RNA-Seq through SOLID System. Results: By analysis of RNA-Seq data and RT-PCR validation we have identified novel transcripts in several MED genes (including MED1, MED 15, MED 17 and MED23). Some of these transcripts are different in their 5’ and 3’ UTRs. Other transcripts are differently spliced thus excluding or including known/new exons. Conclusions: This findings contribute to the characterization of novel MED transcripts in EPCs that could participate to the regulation of genes involved in different cell states. Our findings could have relevant implications in the regenerative action of EPCs in the cardiovascular system immune response after cardiac transplantation.
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COMPARISON OF DIFFERENT EXTRACTION PROTOCOLS ON DNA YEILD AND PURITY. Lucie Richard. Reference and Stem Cell Laboratory, Hema-Quebec, Saint-Laurent, QC, Canada. Aim: DNA extraction is the first step of sample transformation before HLA typing analysis. This manipulation is thought very important and is crucial in terms of yield and purity of the DNA, especially when working with mouth swabs, dry blood from FTA paper, or old blood sample as starting material. An urban legend in our laboratory was that one mouth swab could not give sufficient amount of DNA to allow a low resolution HLA-A, B, C and high resolution DRB1 typing. Another urban legend was suggesting that blood older than 7days must inevitably be extracted manually on a column. We wanted to challenge these popular beliefs. We report here the comparative study we made in order to reorganize our DNA collection strategy. Methods: Three different protocols were used to extract and purify DNA from samples of different origins. The Maxwell 16 system (Promega), the GenoM-6 system (Qiagen) and the manual QiaAmp Column kit (Qiagen) were compared for DNA extraction from mouth swabs, whole blood (either fresh and up to 21 day old), cord blood, buffy coat and dry blood on FTA paper. The DNA collected was first evaluated for quantity and quality and should be sufficient to allow the HLA-A, B, C, DRB1 and DQB1 typing by SSO, SSP and/or SBT methods. The second step was to verify that these analyses could be performed without any problem. Results: The first step of the study revealed that the concentration of DNA extracted from mouth swabs using the Maxwell 16 system was the highest among the different extraction systems and that only one mouth swab was sufficient to generate a complete HLA typing. Also, old blood (as old as 21 days) can be extracted without any problem by any of the 3 methods. The second step of the study demonstrated that the quality and quantity of the DNA collected was suitable for HLA-A, B, C, DRB1 and DQB1 typing using SSO, SSP and SBT techniques. Conclusions: Urban legends were proven wrong and the DNA collection strategy was able to be revised with the collaboration of all the techs.
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Abstracts / Human Immunology 74 (2013) 51–173
IDENTIFICATION BY RNA-SEQ OF NOVEL ANCESTRAL MEDIATOR TRANSCRIPTS IN ENDOTHELIAL PROGENITORS. Monica Rienzo 1, Antonietta Picascia 2, Concetta Schiano 3, Amelia Casamassimi 1, Vincenzo Grimaldi 2, Claudio Napoli 1,2. 1 Department of General Pathology, Division of Clinical Pathology and Excellence Research Centre on Cardiovascular Disease, Second University of Naples, Naples, Italy; 2 U.O.C. Division of Immunohematology, Transfusion Medicine and Transplant Immunology (SIMT), Regional Reference Laboratory of Transplant Immunology (LIT), Second University of Naples, Naples, Italy; 3 Institute of Diagnostic and Nuclear Development, SDN Foundation, Naples, Italy. Aim: Mediator (MED) complex functions as a pivotal adaptor between transcription factors bounded at gene regulatory elements, RNA polymerase II and general transcription factors. Different ancestral humanMED complexes including at least 30 distinct MED subunits (MEDs) have been isolated. Because of the importance of ancestral MED role in the transcription of the eukaryotic genes, disruption of MED function may have relevant pathophysiological consequence also in the cardiovascular system and transplantation. Here, we have analyzed the expression data relative to MEDs in human endothelial progenitor cells (EPCs) obtained by RNA-Seq on a next generation sequencing platform. The introduction of next generation sequencing (NGS) technologies has revealed the complexity of mammalian transcriptomes, enabling to effectively explore, with an unprecedented throughput capacity, simple and complex genomes and even their differences in health and disease conditions. Methods: RNA was isolated from early human EPCs and after ribodepletion it was used for the RNA-Seq through SOLID System. Results: By analysis of RNA-Seq data and RT-PCR validation we have identified novel transcripts in several MED genes (including MED1, MED 15, MED 17 and MED23). Some of these transcripts are different in their 5’ and 3’ UTRs. Other transcripts are differently spliced thus excluding or including known/new exons. Conclusions: This findings contribute to the characterization of novel MED transcripts in EPCs that could participate to the regulation of genes involved in different cell states. Our findings could have relevant implications in the regenerative action of EPCs in the cardiovascular system immune response after cardiac transplantation.
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COMPARISON OF DIFFERENT EXTRACTION PROTOCOLS ON DNA YEILD AND PURITY. Lucie Richard. Reference and Stem Cell Laboratory, Hema-Quebec, Saint-Laurent, QC, Canada. Aim: DNA extraction is the first step of sample transformation before HLA typing analysis. This manipulation is thought very important and is crucial in terms of yield and purity of the DNA, especially when working with mouth swabs, dry blood from FTA paper, or old blood sample as starting material. An urban legend in our laboratory was that one mouth swab could not give sufficient amount of DNA to allow a low resolution HLA-A, B, C and high resolution DRB1 typing. Another urban legend was suggesting that blood older than 7days must inevitably be extracted manually on a column. We wanted to challenge these popular beliefs. We report here the comparative study we made in order to reorganize our DNA collection strategy. Methods: Three different protocols were used to extract and purify DNA from samples of different origins. The Maxwell 16 system (Promega), the GenoM-6 system (Qiagen) and the manual QiaAmp Column kit (Qiagen) were compared for DNA extraction from mouth swabs, whole blood (either fresh and up to 21 day old), cord blood, buffy coat and dry blood on FTA paper. The DNA collected was first evaluated for quantity and quality and should be sufficient to allow the HLA-A, B, C, DRB1 and DQB1 typing by SSO, SSP and/or SBT methods. The second step was to verify that these analyses could be performed without any problem. Results: The first step of the study revealed that the concentration of DNA extracted from mouth swabs using the Maxwell 16 system was the highest among the different extraction systems and that only one mouth swab was sufficient to generate a complete HLA typing. Also, old blood (as old as 21 days) can be extracted without any problem by any of the 3 methods. The second step of the study demonstrated that the quality and quantity of the DNA collected was suitable for HLA-A, B, C, DRB1 and DQB1 typing using SSO, SSP and SBT techniques. Conclusions: Urban legends were proven wrong and the DNA collection strategy was able to be revised with the collaboration of all the techs.