TRANSCRIPTOME 2002: From Functional Genomics to Systems
Clinical Utility of Quantitative Real-Time PCR for Monitoring Leukemia Patients Using RNA Stabilizer In Blood Samples
Ryuji Kawaguchi1, Hideyuki Kudoh1, Yasuhumi Matsuda1, Thoru Murayama2 and Ralf Wyrich3, 1Genetics Research Institute, SRL, Inc., Tokyo, JAPAN, 2Hyogo Medical Centre for Adults, Hyogo, JAPAN, 3QIAGEN GmbH, Hilden, GERMANY
Chimeric mRNA detection is used for minimal residual disease (MRD) monitoring in patients with leukemia. However, degradation of mRNA in patient samples may impede accurate data analysis. We have evaluated a new system for sample collection, RNA stabilization and purification (PAXgene[tm] Blood RNA System, PreAnalytiX GmbH). For this evaluation we used real-time PCR methods, that were optimised to determine BCR-ABL (Major bcr and minor bcr) in CML patients with t(9;22), AML1-MTG8 in AML patients with t(8;21) and PML-RARA in APL patients with t(15;17). Real-time quantitative PCR is done using the LightCycler[tm] (Roche) with hybridisation probes for BCR-ABL, AML1-MTG8, PML-RARA and G6PDH. Each quantification assay is optimised to achieve sensitivity between 10 and 100 copies/[mu]g of total RNA, with dynamic ranges between 1 to 106 dilutions of a positive cell line in a negative cell line. The mRNA quantifications were carried out using blood from normal donors that was spiked with cells from cell lines carrying the genetic markers described above. Expression levels of the house keeping genes, G6PDH, were used as controls. Blood samples from identical donors were collected and stored in PAXgene Blood RNA tubes and EDTA tubes for extended periods of time (day0 - day21). Our data shows significantly higher mRNA detection levels in PAXgene-stabilized samples as compared EDTA blood samples.
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