Assessment of a new RNA stabilizing reagent (Tempus Blood RNA) for minimal residual disease in onco-hematology using the EAC protocol.

Author(s): Prezeau N, Silvy M, Gabert J, Picard C

Publication: Leuk Res, 2006, Vol. 30, Page 569-74

PubMed ID: 16209886 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 stabilization and extraction methods on RNA quality, and quantity. Pooled blood from 20 healthy individuals was divided between 6 replicate PAXgene, Tempus and EDTA tubes. RNA extracted using PAXgene and Tempus was concentrated before being reverse transcribed according to the Europe Against Cancer (EAC) protocol. For determining the sensitivity of detecting BCR-ABL FG transcripts, different percentages of K562 cells carrying the BCR-ABL FG were mixed into the blood.

   Summary of Findings:

   The RNA yield from unstabilized EDTA-blood extracted using Ficoll/Trizol was 9 ug, while 6 ug of RNA were isolated from PAXgene-stabilized and extracted blood and 3 ug were isolated from Tempus-stabilized and extracted blood. The RNA quality, as assessed by bioanalyzer, was similar between the three methods, but a third ribosomal peak (5S) was present in Trizol-extracted specimens, and 20% of Tempus specimens showed slight degradation. While cycle threshold (CT) values depended on the extraction and stabilization methods used, the ratio of the GUS to ABL transcripts were similar between methods. The sensitivity of detection of BCR-ABL FG transcripts was similar between differently extracted specimens.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • None (Fresh)
   • PAXgene

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) PAXgene Tempus
   Analyte Extraction and Purification	Analyte isolation method	PAXgene blood kit Tempus blood RNA kit EDTA blood with Ficoll/Trizol extraction
   Real-time qRT-PCR Specific	Targeted nucleic acid	ABL GUS BCR-ABL

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2. Study Purpose

   The purpose of this study was to determine the effects of storage of blood in Tempus instead of EDTA tubes on RNA yield and recovery of BCR-ABL FG transcripts from spiked specimens. Blood from 3 healthy individuals was mixed with 1% K562 cells carrying the BCR-ABL FG. RNA was extracted from blood in Tempus tubes using the Tempus blood kit and from blood in EDTA tubes using a Ficoll/Trizol method.

   Summary of Findings:

   The RNA yield from EDTA-blood was stable for the first three days of storage at room temperature, but the yield decreased by 50% when EDTA-blood was stored for 5 days at room temperature. In contrast, RNA yield was not affected by storage of blood at room temperature for 5 days in Tempus tubes. Further, specimens stored in EDTA tubes had increased IL8 and decreased BCR-ABL levels after 5 days, but this was not found for specimens stored in Tempus tubes. The ratio of BCR-ABL FG/ABL was stable in blood stored in Tempus tubes for 45 days at room temperature or 4 degrees C or when stored for 6 months at -20 or -80 degrees C. The authors report that storage of blood in Tempus tubes for up to 6 months at any temperature had no effects on RNA yield or 28S/18S rRNA ratio.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Type of storage container	Tempus tube EDTA tube
   Storage	Time at room temperature	0 days 1 day 3 days 5 days
   Storage	Storage temperature	-80 degrees C -20 degrees C 4 degrees C 22 degrees C
   Storage	Storage duration	0 days 45 days 3 months 6 months
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Tempus Frozen Refrigeration
   Real-time qRT-PCR Specific	Targeted nucleic acid	BCR-ABL IL8 ABL 28s/18s rRNA

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