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Gene expression profiling of whole blood: A comparative assessment of RNA-stabilizing collection methods.

Author(s): Donohue DE, Gautam A, Miller SA, Srinivasan S, Abu-Amara D, Campbell R, Marmar CR, Hammamieh R, Jett M

Publication: PLoS One, 2019, Vol. 14, Page e0223065

PubMed ID: 31600258 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared RNA quality and gene expression patterns in RNA extracted from PBMCs preserved in Trizol and from blood stored in PAXgene RNA and RNAgard tubes. Blood was collected from eight healthy army personnel 2 weeks prior to deployment using a 21-gauge butterfly needle and catheter. Blood from each subject was dispensed into a PAXgene RNA tube, a RNAgard Blood tube, and two CPT tubes. PAXgene and RNAgard specimens were inverted 10 times and stored at room temperature for 2 h before freezing at-20°C for 24 h and storage at -80°C until transport. PBMCs were isolated from blood in CPT tubes within 2 h of collection by centrifugation at 1700 x g for 20 min. PBMCs were washed in PBS and resuspended in Trizol. All specimens were then transported on dry ice. Cell pellets were obtained from blood in PAXgene tubes by centrifugation at 3500 x g for 10 min, resuspension in water, and a second centrifugation. Cell pellets were obtained from blood in RNAgard tubes by mixing 4 mL of blood with BioMaxi Precipitation Buffer in a new tube, incubation for 15 min, and then centrifugation at 4500 x g for 30 min. RNA was extracted from the PAXgene and RNAgard pellets using the PAXgene RNA blood miRNA kit following instructions for the QIAcube. RNA was extracted from frozen PBMCs in Trizol following the Trizol protocol. RNA was quantified by spectrophotometry. RNA integrity was evaluated using an Agilent RNA ScreenTape assay. Gene expression was evaluated using the Agilent SurePrint G3 Human gene expression v2 8 × 60K Microarray following the manufacturer’s workflow. Data was normalized and converted to log 2-fold differences and quantile normalized across arrays and other methods using R scripts.

   Summary of Findings:

   Mean RIN values were higher for RNA extracted from PBMCs in Trizol than from blood in PAXgene or RNAgard tubes (8.875 versus 7.306 and 7.433, respectively; P<0.0001), but there was no difference in RIN values between PAXgene and RNAgard samples. While the overall fold-changes in expression appeared similar across the specimen types both before and after normalization, PCA clustered by specimen type rather than subject. The authors point out that the separate clustering of the PBMCs in Trizol versus the two blood specimen types is explained by the different cell type compositions. The variation between technical replicates was lowest in the PBMCs preserved in Trizol, but the separation between subjects was also lower than for the two types of blood specimens. The signal-to-noise ratio (ratio of correlation of within-donor probes to between-donor probes for a given method) was close to 1 for all methods (1.058 for PAXgene, 1.064 for RNAgard, and 1.044 for PBMCs in Trizol) indicating a similar ability of each to differentiate mRNA levels across individuals. Only very small weak correlations were noted between probe properties (distance from 3’, distance from 5’, target size, %GC content) and probe variability for each method, indicating differences between methods are not due to RNA degradation. In PAXgene and RNAgard specimens, the technical variance was highest for genes associated with immune response and homeostasis functions. Expression of nuclear lumen and apoptotic genes was higher and metal ion binding gene lower in RNAgard specimens than PAXgene specimens.

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • PAXgene
   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	CPT tube PAXgene RNA tube RNAgard blood tube
   Biospecimen Preservation	RNA stabilization method	PAXgene tube Trizol reagent RNAgard blood tube
   Biospecimen Aliquots and Components	Blood and blood products	Peripheral blood mononuclear cells Peripheral blood

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