Flow-based sorting of neonatal lymphocyte populations for transcriptomics analysis

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 2.5 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.

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues

“…To ensure consistent instrument performance, the flow cytometer was calibrated with Peak 6 Rainbow Calibration Particles (Spherotech, Lake Forest, IL). A detailed description of calibration methods was previously published (24).…”

“…RNA sequencing. RNA sequencing (RNAseq) was performed on PMX and sorted cells with 1 ng of total RNA per sample amplified using SMARter Ultra Low amplification kit (Clonetech, Mountain View, CA) as described previously (24). Reads were aligned using the Splice Transcript Alignment to a Reference algorithm (12), and expression values were summarized using high-throughput sequencing (1).…”

Association of Dynamic Changes in the CD4 T-Cell Transcriptome With Disease Severity During Primary Respiratory Syncytial Virus Infection in Young Infants

“…Heparinized blood was maintained at room temperature for up to 2 hours, and peripheral blood mononuclear cells were isolated by Ficoll-hypaque gradient, flow-sorted into subsets including CD3 + CD4 + CD8 − lymphocytes, and stored in RNA lysis buffer at −20°C [21].…”

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

“…This highlights that our protocol represents a robust alternative to protein-based assays (e.g., when measuring changes in cytokine mRNA expression in PBMCs in response to specific in vitro stimulation). This work will significantly improve analytical capacity of studies relying on irreplaceable, relatively small or costly human samples (e.g., neonatal PBMCs) (66,67).…”