Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays.

Author(s): Thach DC, Lin B, Walter E, Kruzelock R, Rowley RK, Tibbetts C, Stenger DA

Publication: J Immunol Methods, 2003, Vol. 283, Page 269-79

PubMed ID: 14659918 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 storing blood in PAXgene tubes at room temperature for 9 h followed by -20 degrees C for 6 days rather than for 2 h at room temperature on the RNA transcriptome determined by microarray. After storage, RNA was extracted immediately, and RNA from specimens stored for 2 h was frozen until all samples could be analyzed.

   Summary of Findings:

   Specimens stored for 2 h at room temperature had higher RNA yield, as determined by spectrophotometer (7.3 ug versus 6.3 ug, p=0.007), and more DNA contamination, as determined by real-time PCR of GAPDH, than the specimens stored for 9 h at room temperature followed by 6 days at -20 degrees C. After DNAse treatment, RNA yields were lower, as determined by integration of fluorescence (3.8 ug versus 4.7 ug, p=0.025), in specimens stored for 2 h at room temperature than in the specimens stored for 9 h at room temperature followed by 6 days at -20 degrees C. On column DNAse treatment was not sufficient to remove DNA contamination, but use of in-solution DNAse was, as determined by real-time PCR amplification of GAPDH in RNA. RNA purity and quality, cDNA and cRNA yield, cRNA purity and microarray quality metrics were not affected by storage duration. 99% of the variation in expression of 100 selected genes was based on gene (p<0.001), but 0.17% of the variation was determined by the microarray platform (HGU133a versus HGU133b, p<0.001) and 0.09% of the variation was determined by the storage duration of the specimen in the PAXgene tube (p<0.001), according to ANOVA.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • PAXgene

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	2 h 9 h
   Real-time qPCR Specific	Targeted nucleic acid	GAPDH
   Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH
   Analyte Extraction and Purification	Nucleic acid digestion	None In-solution DNAse On-column DNAse
   DNA microarray Specific	Technology platform	HGU133a HGU133b

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