Differential gene expression profiles are dependent upon method of peripheral blood collection and RNA isolation.

Author(s): Asare AL, Kolchinsky SA, Gao Z, Wang R, Raddassi K, Bourcier K, Seyfert-Margolis V

Publication: BMC Genomics, 2008, Vol. 9, Page 474

PubMed ID: 18847473 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of blood preservation method and RNA extraction method on gene expression analysis from PHA stimulated and unstimulated blood using microarrays. Unless otherwise specified blood was collected into tubes without anticoagulant. Blood was transferred to the PAXgene or Tempus tubes.

   Summary of Findings:

   RNA extraction using the Tempus system resulted in a higher average RNA yield, ratio of absorbance at 260 to 280, and percentage of present calls than extraction using the PAXgene kit, and lower 3' to 5' GAPDH ratio than PAXgene. Hierarchical clustering properly clustered specimens based on PHA stimulation, but a secondary clustering based on extraction method was also observed. 538 genes were upregulated, and 392 genes were downregulated by PHA stimulation when RNA was extracted with Tempus, but when RNA was isolated with PAXgene, 400 genes were upregulated, and 539 were downregulated by PHA stimulation. 56% of the upregulated genes and 40% of the downregulated genes showed similar directional changes for both extraction kits. A number of immune transcripts were confirmed to be differentially expressed after PHA stimulation using PCR with MALDI-TOF mass spectrometry, but in PAXgene extracted specimens, variability precluded statistical significance. Collection of specimens into lithium heparin tubes resulted in changes in the expression of B cell translocation transcript compared to collection in tubes without anticoagulant.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • PAXgene

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	MALDI-TOF MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Tempus PAXgene
   Biospecimen Acquisition	Anticoagulant	Lithium heparin None
   Biospecimen Aliquots and Components	Biospecimen components	0 ug/ml PHA 25 ug/ml PHA
   Biospecimen Preservation	Type of fixation/preservation	PAXgene Tempus

   View more