Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays.
Genome-wide expression studies of human blood samples in the context of epidemiologic surveillance are confronted by numerous challenges-one of the foremost being the capability to produce reliable detection of transcript levels. This led us to consider the Paxgene Blood RNA System, which consists of a stabilizing additive in an evacuated blood collection tube (PAX tube) and a sample processing kit (PAX kit). The PAX tube contains a solution that inhibits RNA degradation and gene induction as blood is drawn into the tube. The stability of RNA in PAX tubes under conditions for practical clinical applications has been determined by RT-PCR, but has not been assessed at the transcriptome level on Affymetrix microarrays. Here, we report a quality assured and controlled protocol that is capable of producing reliable gene expression profiles using the GeneChip system with RNA isolated from PAX tubes. Using this protocol, we compared quality metrics and gene-expression profiles of RNA, extracted from blood in PAX tubes that sat at room temperature for 2 h, with that of blood in PAX tubes incubated at room temperature for 9 h followed by storage at -20 degrees C for 6 days. Of numerous metrics, differences between the two handling methods were detected for the level of DNA contamination, RNA yield, and double stranded cDNA yield. Analysis of variance of gene-expression revealed small but significant differences between the handling methods. These results contribute to the determination of protocols for clinical studies and progress us towards the goal of using the transcriptome in diagnosis and surveillance.
Thach
Dzung C
DC
Center for Biomolecular Science and Engineering, Code 6910, Naval Research Laboratory, 4555 Overlook Avenue SW, Building 30, Washington, DC 20375, USA.
Lin
Baochuan
B
Walter
Elizabeth
E
Kruzelock
Rusty
R
Rowley
Robb K
RK
Tibbetts
Clark
C
Stenger
David A
DA
D001800:Blood Specimen Collection / Q000379:methods*; D020869:Gene Expression Profiling; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000097:blood*
D012313:RNA