Implementation of a proficiency testing for the assessment of the preanalytical phase of blood samples used for RNA based analysis.

Author(s): Günther K, Malentacchi F, Verderio P, Pizzamiglio S, Ciniselli CM, Tichopad A, Kubista M, Wyrich R, Pazzagli M, Gelmini S

Publication: Clin Chim Acta, 2012, Vol. 413, Page 779-86

PubMed ID: 22285774 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 PAXgene blood stabilization and a 2 h delay in RNA extraction on RNA integrity and expression. After preparation, RNA was shipped on dry ice. RNA was isolated from specimens in PAXgene tubes using PAXgene system and from specimens in EDTA tubes by a standard protocol (phenol and or column based protocols).

   Summary of Findings:

   Much lower minimum RIN were observed in RNA obtained from specimens collected in EDTA tubes than from specimens collected in PAXgene tubes. While the median RIN for RNA obtained from specimens collected in EDTA or PAXgene tubes and not stored or in PAXgene tubes and stored for 2 h at room temperature was >8, the median RIN in RNA from specimens stored for 2 h at room temperature in EDTA tubes was 3.1 and only 2/7 had RIN >8. The effect of delayed extraction of RNA from PAXgene blood on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), interleukin (IL)8, IL1B, and FBJ murine osteosarcoma viral oncogene homolog (FOS) was small, but differences in expression of GAPDH, IL8, IL1B and FOS between RNA from EDTA tubes that were extracted immediately rather than stored at room temperature for 2 h before extraction were large and highly variable.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 h 2 h
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) PAXgene
   Real-time qRT-PCR Specific	Targeted nucleic acid	IL1B IL8 FOS GAPDH
   Biospecimen Acquisition	Type of collection container/solution	PAXgene tube EDTA tube

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2. Study Purpose

   The purpose of the study was to determine the effect of PAXgene blood stabilization and overnight shipment of blood or RNA on RNA purity, integrity and expression in blood specimens. The initial specimen was collected in EDTA tubes and aliquoted into PAXgene tubes. RNA was extracted immediately and shipped to the analyzing lab or to the intermediate lab, or blood was shipped at ambient temperatures overnight to the intermediate lab before RNA extraction. RNA was shipped on dry ice from the initial lab or the intermediate lab to the SPIDIA lab.

   Summary of Findings:

   No significant differences in RNA purity, RIN, yield or expression were observed between RNA analyzed after shipment once or twice on dry ice. When whole blood was shipped overnight at ambient temperatures the median RIN numbers were >8 in specimens shipped in PAXgene and EDTA tubes, but two of nine specimens shipped in EDTA tubes had RIN <7, and overall, there was more variability in RIN, purity and yield from specimens shipped in EDTA tubes than PAXgene tubes. The levels of GAPDH were equal, IL1B tended to be lower, and IL8 and FOS levels tended to be higher in specimens shipped in EDTA tubes than those shipped in PAXgene tubes, and there was more variability in the expression of all genes when specimens were shipped in EDTA tubes than when shipped in PAXgene tubes. Further, the authors report that generally, RNA shipped in PAXgene tubes had expression levels that were closer to those in unshipped specimens than specimens shipped in EDTA tubes.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) PAXgene
   Storage	Between site transportation method	Mailed Not transported Transported once Transported twice
   Storage	Time at room temperature	0 h 2 h
   Real-time qRT-PCR Specific	Targeted nucleic acid	IL1B IL8 FOS GAPDH

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