Assessment of a new RNA stabilizing reagent (Tempus Blood RNA) for minimal residual disease in onco-hematology using the EAC protocol.
Standardization efforts of real time quantitative PCR (RQ-PCR), such as the Europe Against Cancer (EAC) protocol, are actually essential. However, all the stages of the preanalytical phase (blood collection, conservation and procedures of cellular separation) which influence the ex vivo expression of many genes are not controlled. Various kits for stabilizing the RNA at the time of blood collection were developed: PAXgene Blood kit (Qiagen) and the new Tempus Blood RNA kit (Applied Biosystems). PAXgene Blood was already validated to monitor minimal residual disease (MRD) in onco-hematologic pathologies. In order to evaluate the Tempus Blood RNA kit, it was compared to unstabilized EDTA blood/Ficoll/TRIzol protocol and PAXgene Blood kit. The RNAs extracted by the different methods were assessed for quantity, quality and detection of control genes (GUS and ABL) and fusion gene transcripts BCR-ABL and TEL-AML1 by RQ-PCR. Our study shows that using the EAC protocol, the new Tempus Blood RNA kit allows the detection of fusion gene transcript with the same sensitivity as the two other protocols. Altogether, our data suggest the possible use of such a technology for MRD follow-up in myeloproliferative diseases and acute leukemias. So, further multicentric studies on leukemic patients should be performed to validate this technique in these applications.
Prezeau
Nicolas
N
Department of Biochemistry & Molecular Biology, Hôpital Universitaire Nord, ERT-MEIDIA, IFR Jean Roche, Université de la Méditerranée, Marseille, France.
Silvy
Monique
M
Gabert
Jean
J
Picard
Christophe
C
D045744:Cell Line, Tumor; D005060:Europe; D019337:Hematologic Neoplasms / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D020014:K562 Cells; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D012313:RNA / Q000032:analysis*; D020871:RNA Stability; D011933:Reagent Kits, Diagnostic / Q000592:standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D007202:Indicators and Reagents; D015514:Oncogene Proteins, Fusion; D011933:Reagent Kits, Diagnostic; D012313:RNA