title: Next-generation qPCR for the high-throughput measurement of gene expression in multiple leukocyte subsets.
abstract: Clinical studies of gene expression are increasingly using the whole blood, peripheral blood mononuclear cells, and leukocyte subsets involved in the innate and adaptive immune responses. However, the small amount of RNA available in the clinical setting is a limitation for commonly used methods such as quantitative polymerase chain reactions (qPCR) and microarrays. Our aim was to design 96 gene assays to simultaneously measure gene expression in the whole blood and seven leukocyte subsets using a new-generation qPCR method--high-throughput nanofluidic reverse transcription qPCR (HT RT-qPCR). The leukocyte subset purity was 94% to 98% for seven subsets and was less for the γδ T-cell receptor subset (80%). The HT RT-qPCR replicate sample measurements were highly reproducible (r = 0.997, p < 2.2 × 10(-16)), and the ΔΔCt values from HT RT-qPCR correlated significantly with those from qPCR. The control genes were differentially expressed across the eight leukocyte subsets in the control subjects (p = 1.3 × 10(-5), analysis of variance). Two analytical methods, absolute and relative, gave concordant results and were significantly correlated (p = 1.9 × 10(-9)). HT RT-qPCR permits the rapid, reproducible, and quantitative measurement of multiple transcripts using minimal sample amounts. The protocol described yielded leukocyte subsets of high purity and identified two analytic methods for use.
authors.affiliation: 1Department of Neurology, SUNY Downstate Medical Center, Brooklyn, NY, USA.
authors.forename: Mateusz G
authors.initials: MG
authors.lastname: Adamski
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Erin
authors.initials: E
authors.lastname: Wagner
authors.forename: Hua
authors.initials: H
authors.lastname: Yu
authors.forename: Chloe
authors.initials: C
authors.lastname: Seales-Bailey
authors.forename: Steven A
authors.initials: SA
authors.lastname: Soper
authors.forename: Michael
authors.initials: M
authors.lastname: Murphy
authors.forename: Alison E
authors.initials: AE
authors.lastname: Baird
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000378:metabolism*; D016022:Case-Control Studies; D005260:Female; D015870:Gene Expression; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D006801:Humans; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D020521:Stroke / Q000097:blood / Q000235:genetics* / Q000473:pathology; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger
keywords: gene expression; leukocyte subsets; next-generation qPCR; normalization; reference genes