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Tytuł pozycji:

Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis.

Tytuł:
Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis.
Autorzy:
Misra RS; Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States.
Bhattacharya S; Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States; Pediatric Molecular and Personalized Medicine Program, University of Rochester Medical Center, Rochester, NY 14642, United States.
Huyck HL; Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States.
Wang JC; Rochester Human Immunology Center, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, United States.
Slaunwhite CG; Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States.
Slaunwhite SL; Shared Resources Laboratories, University of Rochester Medical Center, Rochester, NY 14642, United States.
Wightman TR; Shared Resources Laboratories, University of Rochester Medical Center, Rochester, NY 14642, United States.
Secor-Socha S; Rochester Human Immunology Center, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, United States.
Misra SK; Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States.
Bushnell TP; Shared Resources Laboratories, University of Rochester Medical Center, Rochester, NY 14642, United States.
Reynolds AM; Department of Pediatrics, University at Buffalo, Buffalo, NY 14222, United States.
Ryan RM; Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, United States.
Quataert SA; Rochester Human Immunology Center, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, United States.
Pryhuber GS; Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States; Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY 14642, United States.
Mariani TJ; Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States; Pediatric Molecular and Personalized Medicine Program, University of Rochester Medical Center, Rochester, NY 14642, United States.
Źródło:
Journal of immunological methods [J Immunol Methods] 2016 Oct; Vol. 437, pp. 13-20. Date of Electronic Publication: 2016 Jul 18.
Typ publikacji:
Journal Article; Research Support, N.I.H., Extramural
Język:
English
Imprint Name(s):
Publication: Amsterdam : Elsevier
Original Publication: Amsterdam, North-Holand,
MeSH Terms:
Gene Expression Profiling/*methods
Lymphocytes/*physiology
Cell Separation ; Centrifugation, Density Gradient ; Feasibility Studies ; Ficoll ; Flow Cytometry ; High-Throughput Nucleotide Sequencing ; Humans ; Infant, Newborn ; Lymphocyte Count ; Miniaturization
References:
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Grant Information:
HHSN272201200005C United States AI NIAID NIH HHS; U01 HL101794 United States HL NHLBI NIH HHS; U01 HL101813 United States HL NHLBI NIH HHS; U01 HL122700 United States HL NHLBI NIH HHS
Contributed Indexing:
Keywords: BPD; Flow sorting; Lymphocytes; PBMC; Prematurity; RNASeq; T-Cell
Substance Nomenclature:
25702-74-3 (Ficoll)
Entry Date(s):
Date Created: 20160721 Date Completed: 20170705 Latest Revision: 20181113
Update Code:
20240104
PubMed Central ID:
PMC5247270
DOI:
10.1016/j.jim.2016.07.001
PMID:
27438473
Czasopismo naukowe
Rationale: Emerging data suggest an important role for T lymphocytes in the pathogenesis of chronic lung disease in preterm infants. Comprehensive assessment of the lymphocyte transcriptome may identify biomarkers and mechanisms of disease.
Methods: Small volume peripheral blood samples were collected from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of discharge from the hospital. Blood samples were collected at two sites and shipped to a central laboratory for processing. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation and separated into individual lymphocyte cell types by fluorescence-activated cell sorting. Gating strategies were optimized to ensure reproducible recovery of highly purified lymphocyte populations over a multi-year recruitment period. RNA was isolated from sorted cells and characterized by high-throughput sequencing (RNASeq).
Results: Blood volumes averaged 2.5ml, and sufficient PBMCs were collected from 165 of the 246 samples obtained (67%) from the 277 recruited subjects to complete sorting and RNASeq analysis on the resulting sorted cells. The number of total lymphocytes per ml of blood in the neonatal subjects was approximately 4 million/ml. Total lymphocyte frequencies recovered following sort varied widely among subjects, as did the frequency of individual lymphocyte and NK cell sub-populations. RNA yield from sorted cells varied according to cell type, but RNA of sufficient quantity and quality was recovered to enable RNASeq.
Summary: Our results describe a validated procedure for the generation of genome-wide expression data from isolated lymphocyte sub-populations obtained from newborn blood.
(Copyright © 2016. Published by Elsevier B.V.)

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