Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis
Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis
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DOI：
10.1016/j.jim.2016.07.001
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发表时间：
2016-10-01
影响因子：
2.2
通讯作者：
Mariani, Thomas J.
中科院分区：
文献类型：
--
作者：
Misra, Ravi S.;Bhattacharya, Soumyaroop;Mariani, Thomas J.
原理：新的数据表明T淋巴细胞在早产儿慢性肺部疾病的发病机制中起重要作用。淋巴细胞转录组的综合评估，可以确定生物标志物和疾病mechanisms of disease.Methods：小体积外周血样本收集自早产儿同意参加早产儿和呼吸系统结局计划（PROP），在出院时。在两个研究中心采集血样，并运送至中心实验室进行处理。通过Ficoll-Hypaque梯度离心分离外周血单核细胞（PBMC），并通过荧光激活细胞分选分离成单个淋巴细胞类型。优化了门控策略，以确保在多年的招募期内高度纯化的淋巴细胞群的可重复回收。结果：从277例受试者的246个样本中，平均血容量为2.5 ml，有165个样本（67%）采集到足够的PBMC，完成了分选和RNASeq分析。新生儿受试者中每ml血液中的总淋巴细胞数量约为4百万/ml。分类后恢复的总淋巴细胞频率在受试者之间差异很大，单个淋巴细胞和NK细胞亚群的频率也是如此。从分选细胞的RNA产量根据细胞类型而变化，但足够的数量和质量的RNA被回收，使RNASeq.Summary：我们的结果描述了一个验证的程序，从新生儿血液中获得的分离的淋巴细胞亚群的全基因组表达数据的生成。(C)2016由Elsevier B. V.出版
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 2.5 ml, 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. (C) 2016 Published by Elsevier B.V.