Peripheral blood gene expression: it all boils down to the RNA collection tubes.
Author(s): Menke A, Rex-Haffner M, Klengel T, Binder EB, Mehta D
Publication: BMC Res Notes, 2012, Vol. 5, Page 1
PubMed ID: 22214347 PubMed Review Paper? No
Purpose of Paper
This paper compared RNA yields, purity, integrity and gene expression between blood specimens collected in PAXgene and Tempus tubes from healthy men and investigated the effects of globin reduction and dexamethasone stimulation.
Conclusion of Paper
Although less RNA was obtained from blood collected in PAXgene tubes than Tempus tubes, both tubes yielded RNA with RNA integrity numbers (RIN) >8.0. Gene expression levels were strongly correlated between tube types, although a slightly higher but nonsignificant mean number of transcripts were detected in RNA isolated from Tempus than PAXgene blood. For both PAXgene and Tempus blood specimens, globin reduction significantly decreased RNA yield, and average RIN, but significantly increased the number of transcripts with signal above the detection threshold and decreased the mean variance in expression.
While transcripts that were differentially expressed after dexamethasone stimulation were detected in both PAXgene and Tempus blood specimens, the majority of transcripts were only detectable in one tube type. Pathway analysis showed dexamethasone stimulation led to enrichment of genes in the Toll receptor, adipogenesis, T-cell receptor, and IL-7 and IL- 2 pathways in both specimen types; in the DNA damage response and cell cycle pathways in Tempus blood specimens; and in translation factors, pentose phosphate, TGF-beta receptor and Hedgehog signaling pathways in PAXgene blood specimens.
Studies
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Study Purpose
This study compared RNA yields, purity, integrity and gene expression between blood specimens collected in PAXgene and Tempus tubes from healthy men and investigated the effects of globin reduction and dexamethasone stimulation. Blood was collected into PAXgene and Tempus blood tubes from three healthy men following a 2 h fast and then again 3 h after oral administration of dexamethasone. Blood was stored at room temperature for 2 h before transfer to -20°C. RNA was extracted from blood specimens using tube-specific RNA extraction kits (PAXgene Blood RNA Kit and Tempus Spin RNA Isolation Kit). RNA aliquots were subjected to globin reduction with the GLOBINclear-Human Kit. RNA purity and integrity were assessed by spectrophotometer and bioanalyzer, respectively. Before expression analysis using Illumina HumanHT-12 Expression BeadChips, RNA was amplified using the TotalPrep RNA Amplification Kit. Changes in levels of FKBP5 and IL1SR1 were validated by real-time PCR.
Summary of Findings:
Less RNA was obtained from blood collected in PAXgene tubes than Tempus tubes (2.04 µg/mL versus 2.98 µg/mL, P=0.022). However, RNA obtained from PAXgene and Tempus blood tubes were of comparable purity (average A260/A280 ratios of 2.1, both) and were of high quality (RIN values of 8.7 and 8.2, respectively). Although a slightly higher mean number of transcripts were in RNA isolated from Tempus than PAXgene blood (9265 ± 143 versus 8870 ± 197), the difference was not significant, there was no difference in mean variance, and expression levels were very strongly correlated (r=0.94). Globin reduction led to loss of 47.3% of RNA from PAXgene blood and 27% of RNA from Tempus blood and a significant decrease in average RIN (from 8.7 to 8.2 in PAXgene, P=4.9 x 10-3; and from 8.2 to 7.1 in Tempus, P=4.0 x 10-5). Globin reduction significantly increased the number of transcripts with signal above the detection threshold (from 9762 to 11480 in PAXgene, P=8.17 x 10-5; and from 9918 to 11608 in Tempus, P=1.95 x 10-3) and decreased the mean variance in expression (from 3.2 to 2.89 in PAXgene, P=0.0025; and from 3.21 to 2.94 in Tempus, P=0.041); nevertheless a strong correlation was observed between levels of the 7578 transcripts detected both before and after globin reduction (R=0.97).
After stimulation with dexamethasone, 11107 and 10135 transcripts were detected in PAXgene and Tempus blood specimens, respectively. Of the transcripts found in all specimens regardless of dexamethasone stimulation, 449 and 365 were found to be differentially expressed (fold change ≥ 1.5, adjusted P<0.05) after dexamethasone treatment in PAXgene and Tempus blood specimens, respectively, of which 100 were differentially regulated in both specimen types. When the threshold for differential expression was increased to ≥2 fold change, 31 transcripts in PAXgene and 18 in Tempus specimens were differentially expressed following dexamethasone stimulation, of which only 7 were differentially expressed in both specimen types. Globin reductions did not have a significant effect on the number of differentially expressed transcripts identified. The observed increase in levels of FKBP5 and IL1SR1 following dexamethasone stimulation were validated by real-time PCR. Pathway analysis revealed that dexamethasone stimulation led to enrichment of genes in the Toll receptor, adipogenesis, T-cell receptor, and IL-7 and IL- 2 pathways in both specimen types; in the DNA damage response and cell cycle pathways in Tempus blood specimens; and in translation factors, pentose phosphate, TGF-beta receptor and Hedgehog signaling pathways in PAXgene blood specimens.
Biospecimens
Preservative Types
- PAXgene
- Frozen
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA DNA microarray RNA Spectrophotometry RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution PAXgene tube
Tempus tube
DNA microarray Specific Template modification Globin reduction
No globin reduction
Biospecimen Preservation Type of fixation/preservation PAXgene
Tempus
Preaquisition Other drugs Dexamethasone
None