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J Immunol Methods. 2016 Oct;437:13-20. doi: 10.1016/j.jim.2016.07.001. Epub 2016 Jul 18.

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

Author information

1
Department of Pediatrics, Division of Neonatology, University of Rochester Medical Center, Rochester, NY 14642, United States.
2
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.
3
Rochester Human Immunology Center, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, United States.
4
Shared Resources Laboratories, University of Rochester Medical Center, Rochester, NY 14642, United States.
5
Department of Pediatrics, University at Buffalo, Buffalo, NY 14222, United States.
6
Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, United States.
7
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.

Abstract

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.

KEYWORDS:

BPD; Flow sorting; Lymphocytes; PBMC; Prematurity; RNASeq; T-Cell

PMID:
27438473
PMCID:
PMC5247270
DOI:
10.1016/j.jim.2016.07.001
[Indexed for MEDLINE]
Free PMC Article

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