Preanalytical aspects of quantitative TaqMan real-time RT-PCR: Applications for TF and VEGF mRNA quantification

Author(s): Isaksson Helena S, Nilsson Torbjorn K

Publication: Clin Biochem, 2006, Vol. 39, Page 373

PubMed ID: 16546153 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   To determine whether the type of blood collection tube (PAXgene tube, K3EDTA tube, CPT tube, and EndoTube) affects the quality of extracted RNA.

   Summary of Findings:

   PAXgene collection tubes yielded the highest quality RNA, with distinct 28S and 18S rRNA peaks. While rRNA bands were observed in samples collected in EDTA tubes, degradation was apparent. CPT and Endo tubes were not analyzed.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	PAXgene tube K3EDTA tube CPT tube Endotoxin-free Endotube
   Biospecimen Preservation	Type of fixation/preservation	PAXgene None (fresh)

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2. Study Purpose

   To determine whether the type of blood collection tube adversely impacts TF and VEGF mRNA expression levels.

   Summary of Findings:

   TF and VEGF mRNA levels were significantly lower in samples collected in PAXgene tubes compared to other tube types examined. TF and VEGF expression levels in samples collected in EDTA, CPT, and Endo tubes were comparable.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	PAXgene tube K3EDTA tube CPT tube Endotoxin-free Endotube
   Real-time qRT-PCR Specific	Targeted nucleic acid	TF VEGF
   Biospecimen Preservation	Type of fixation/preservation	PAXgene None (fresh)

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3. Study Purpose

   To determine if a processing delay of 24 h at ambient temperature impacts TF and VEGF mRNA expression levels, and whether results are dependent on collection tube type.

   Summary of Findings:

   Gene expression was not significantly altered in samples stored at ambient temperature for 24 h in PAXgene tubes. All other tube types examined displayed a significant change in expression of one or more of the examined genes (TF, VEGF, beta-actin) after 24 h of storage at ambient temperature.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	K3EDTA tube CPT tube Endotoxin-free EndoTube PAXgene tube
   Storage	Time at room temperature	1 hr 24 hr
   Real-time qRT-PCR Specific	Targeted nucleic acid	TF VEGF beta-actin
   Biospecimen Preservation	Type of fixation/preservation	PAXgene None (fresh)

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