Genome-wide comparison of two RNA-stabilizing reagents for transcriptional profiling of peripheral blood.

Author(s): Nikula T, Mykkänen J, Simell O, Lahesmaa R

Publication: Transl Res, 2013, Vol. 161, Page 181-8

PubMed ID: 23138105 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 using the PAXgene or TEMPUS blood preservation systems, globin reduction, and pre-amplification on RNA yield, quality and transcriptional profiles by Illumina BeadChip Arrays. Blood was processed according to the manufacturers' instructions for each preservation tube type and stored at -70 degrees C. RNA was isolated from PAXgene blood using PAXgene tubes and from TEMPUS blood using the ABI Prism Nucleic Acid PrepStation kit. To investigate the effects of amplification some RNA specimens were amplified with the RiboAmp kit prior to labeling and additional amplification with the Illumina RNA Amplification Kit.

   Summary of Findings:

   While high quality RNA was obtained when either blood preservation system was used, less RNA was obtained from PAXgene blood than TEMPUS blood (1.0 ug/mL blood versus 1.83 ug/mL blood). Globin reduction led to reduced RNA quality, but amplification with RiboAmp did not amplify globin, resulting in reduction of globin compared to other transcripts without RNA degradation. A total of 5200 genes were detected by Illumina BeadChip Array, and 3,058 of these genes were detected in PAXgene, TEMPUS, and frozen blood specimens. The gene expression profile of PBMC was closest to that of blood collected in TEMPUS tubes (r=0.83) rather than blood collected in PAXgene tubes (r=0.75). The Pearson correlations for TEMPUS and PAXgene specimens drawn simultaneously were very strong (r ≥0.94). A total of 443 genes were differentially expressed between PAXgene and TEMPUS specimens, of which 94% were expressed at higher levels in the TEMPUS specimen than the PAXgene specimen. No particular pathways were identified as being affected by the type of preservation system using GSEA, however, ingenuity pathways analysis identified 9 pathways differentially affected by PAXgene and TEMPUS preservation. The differentially expressed genes were enzymes, kinases or transcriptional regulators.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	PAXgene Tempus Frozen
   Biospecimen Acquisition	Type of collection container/solution	PAXgene tube TEMPUS tube
   DNA microarray Specific	Data handling	GSEA Ingenuity pathways analysis
   DNA microarray Specific	Nucleic acid amplification	Preamplified with RiboAmp
   DNA microarray Specific	Template modification	Globin-reduced None
   Analyte Extraction and Purification	Analyte isolation method	ABI Prism Nucleic Acid PrepStation PAXgene spin columns

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