Profiling gene expression in whole blood samples following an in-vitro challenge.
Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.
Spijker
Sabine
S
Department of Molecular and Cellular Neurobiology, Vrije Universiteit, Amsterdam, the Netherlands. sabine.spijker@falw.vu.nl
van de Leemput
Joyce C H
JC
Hoekstra
Chantal
C
Boomsma
Dorret I
DI
Smit
August B
AB
D000328:Adult; D019992:Databases as Topic; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D016247:Information Storage and Retrieval / Q000592:standards; D008297:Male; D012313:RNA / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014429:Twins, Dizygotic / Q000235:genetics*
D012313:RNA