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.

Expression profile based gene clusters for ischemic stroke detection

“…Two variants of CD14 were studied to give a total of 41 transcripts that were tested. The complete primer characteristics were published earlier[18]. The RT-qPCR primers were self-designed, commercially synthesized by Invitrogen and wet tested using regular RT-qPCR (StepOnePlus Real-Time PCR Systems; Applied Biosystems).…”

“…We have previously presented graphical results, based only on Cq values normalized to cell count, of four stroke related transcripts in a cohort of hemorrhagic and ischemic stroke patient [18]. …”

“…These methods, known as high throughput RT-qPCR (HT RT-qPCR) or nanofluidic qPCR, permit the rapid quantification of multiple transcripts using small sample volumes[17,18] with very high sensitivity. Plates can contain up to 96 samples in which 96 transcripts can be simultaneously studied in 9,216 reactions.…”

Recent and near-future advances in nucleic acid-based diagnosis of stroke

“…In a prior study, control gene expression was not stable across leukocyte subsets [66]. Hence, we have developed a method for quantitative analysis of samples using input sample quantity that is independent of control genes [70].…”

“…These methods, known as high-throughput RT-qPCR (HT RTqPCR) or nanofluidic qPCR, permit the rapid quantification of multiple transcripts using small sample volumes [65,66] with very high sensitivity. Plates can contain up to 96 samples in which 96 transcripts can be simultaneously studied in 9216 reactions.…”