Recovery of microarray-quality RNA from frozen EDTA blood samples.

Author(s): Beekman JM, Reischl J, Henderson D, Bauer D, Ternes R, Peña C, Lathia C, Heubach JF

Publication: J Pharmacol Toxicol Methods, 2009, Vol. 59, Page 44-9

PubMed ID: 19028589 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 delayed freezing, processing, and extraction methods on RNA quantity and quality and microarray analysis of EDTA whole blood. RNA obtained from EDTA blood that was frozen immediately after collection, thawed 2 h on ice, and extracted using the QIAamp kit (standard protocol) or PAXgene kit (protocol A), or obtained from EDTA blood that was stored at room temperature for 3 h prior to freezing, and processed like protocol A (protocol B) was compared to RNA obtained from blood collected into a Paxgene tube, frozen immediately, and thawed for 16-21 h at room temperature prior to extraction using the Paxgene kit (reference protocol).

   Summary of Findings:

   When RNA was extracted using the standard protocol, the average yield was 0.07 ug/mL, while an average of 2.84 ug/mL was obtained using the reference protocol. This low yield was not always sufficient, even when pooled, to determine the RNA integrity number (RIN) or to use for microarray analysis. Average RNA yields of 1.76 and 1.29 ug/mL were obtained using protocols A and B, respectively. RINs were lower in RNA obtained by protocols A (6.1) and B (6.4) than RNA obtained using the reference protocol (9.8). When RNA was extracted using the reference protocol, the lowest GAPDH 3 prime to 5 prime ratios, scaling factor and interindividual variability, and the highest percent present calls were obtained. In contrast, when RNA was extracted using the standard protocol, the highest GAPDH 3 prime to 5 prime ratios, and scaling factor, and the lowest percent present calls were obtained. RNA obtained by protocols A and B performed similarly, and was more like RNA obtained by the reference protocol than like RNA obtained by the standard protocol. The call concordance percentages between the specimens obtained using the reference protocol and either the standard protocol, protocol A or protocol B were 77.1%, 87.5% and 85.2%, respectively.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Paxgene tube Potassium EDTA tube
   Storage	Thaw duration	2 h 16-21 h
   Storage	Thaw temperature/condition	On ice Room temperature
   Analyte Extraction and Purification	Analyte isolation method	PAXgene kit QIAamp kit
   Storage	Time at room temperature	0 h 3 h
   Biospecimen Preservation	Type of fixation/preservation	Frozen PAXgene

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