Clinica chimica acta; international journal of clinical chemistry · 2004
A competitive nucleic acid sequence-based amplification assay for the quantification of human MDR1 transcript in leukemia cells.
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Hayashi Tsukasa, Kobayashi Hiroyuki, Miyachi Hayato, Ohshima Toshio, Ujiiye Takeshi, Kawase Masako, Hotta Tomomitsu, Takemura Yuzuru
Аннотация
BACKGROUND: Because clinical drug resistance is caused by low-grade expression of a responsible gene, highly sensitive methods are desirable for its detection in clinical settings. We developed a quantitative nucleic acid sequence-based amplification (NASBA) assay for multidrug resistance-1 (MDR1) transcripts, and applied it to clinical samples.
METHOD: MDR1 transcripts were amplified using the NASBA technique combined with sandwich hybridization of amplified MDR1 mRNA followed by chemiluminescence detection on an automated analyzer. Quantification of MDR1 mRNA was achieved through competitive coamplification of in vitro-generated RNA, which acts as an internal control.
RESULTS: The competitive NASBA assay exhibited higher sensitivity (reliable detection limit was 100 copies of MDR1 mRNA) and linearity over a broader dynamic range (7 logarithmic orders) than the competitive reverse transcription-polymerase chain reaction assay. All 33 clinical samples obtained from patients with leukemia were successfully assayed, demonstrating its feasibility. MDR1 expression-compensated with beta-actin expression-ranged from 1.4 x 10(2) to 2.5 x 10(6) (median 4.8 x 10(5)) copies/microg RNA, while the range of MDR1 expression in peripheral blood samples from 15 healthy adults was from 8.9 x 10(4) to 5.2 x 10(5) (median 2.2 x 10(5)) copies/microg RNA. MDR1 expression in 8 of 33 clinical samples exceeded the median of healthy adult samples.
CONCLUSIONS: The competitive NASBA assay is applicable to MDR1 mRNA quantification in clinical samples and would contribute to detection of clinical multidrug resistance.
METHOD: MDR1 transcripts were amplified using the NASBA technique combined with sandwich hybridization of amplified MDR1 mRNA followed by chemiluminescence detection on an automated analyzer. Quantification of MDR1 mRNA was achieved through competitive coamplification of in vitro-generated RNA, which acts as an internal control.
RESULTS: The competitive NASBA assay exhibited higher sensitivity (reliable detection limit was 100 copies of MDR1 mRNA) and linearity over a broader dynamic range (7 logarithmic orders) than the competitive reverse transcription-polymerase chain reaction assay. All 33 clinical samples obtained from patients with leukemia were successfully assayed, demonstrating its feasibility. MDR1 expression-compensated with beta-actin expression-ranged from 1.4 x 10(2) to 2.5 x 10(6) (median 4.8 x 10(5)) copies/microg RNA, while the range of MDR1 expression in peripheral blood samples from 15 healthy adults was from 8.9 x 10(4) to 5.2 x 10(5) (median 2.2 x 10(5)) copies/microg RNA. MDR1 expression in 8 of 33 clinical samples exceeded the median of healthy adult samples.
CONCLUSIONS: The competitive NASBA assay is applicable to MDR1 mRNA quantification in clinical samples and would contribute to detection of clinical multidrug resistance.
Предметные рубрики MeSH
ATP Binding Cassette Transporter, Subfamily B, Member 1ActinsHumansLeukemiaLeukemia, Myelogenous, Chronic, BCR-ABL PositiveLeukemia, Myeloid, AcuteLuminescent MeasurementsLymphocytesMyelodysplastic SyndromesRNA, MessengerReceptors, TransferrinReverse Transcriptase Polymerase Chain ReactionSelf-Sustained Sequence ReplicationSensitivity and SpecificityTumor Cells, Cultured