title: A novel technology for multiplex gene expression analysis directly from whole blood samples stabilized at ambient temperature using an RNA-stabilizing buffer.
abstract: We describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 μL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 μL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. Gene expression measured on stabilized blood samples was highly correlated (Spearman rank correlation method, ρ = 0.80) to gene expression results obtained with RNA isolated from matched samples (three donors, five technical replicates). Gene expression changes determined with seven radiation-responsive genes on six healthy donor blood samples before and after ex vivo irradiation were highly correlated (ρ = 0.93) to those measured with a TaqMan quantitative real-time RT-PCR assay on RNA purified from matched samples. Thus, this method is reproducible, sensitive, and correlated to quantitative real-time RT-PCR and may be used to streamline the multiplex gene expression analysis of large numbers of stabilized blood samples without RNA purification.
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Chang Hee
authors: CH
authors: Kim
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Majid
authors: M
authors: Abedi
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Yenbou
authors: Y
authors: Liu
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Sree
authors: S
authors: Panuganti
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Francisco
authors: F
authors: Flores
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Kevin R
authors: KR
authors: Shah
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Hannah
authors: H
authors: Catterall
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California.
authors: Krishna S
authors: KS
authors: Morampudi
authors: Department of Research and Development, DxTerity Diagnostics, Rancho Dominguez, California. Electronic address: bob@dxterity.com.
authors: Robert
authors: R
authors: Terbrueggen
mesh_terms: D002021:Buffers; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry*; D020871:RNA Stability; D013696:Temperature
chemical_list: D002021:Buffers; D012313:RNA