A competitive nucleic acid sequence-based amplification assay for the quantification of human MDR1 transcript in leukemia cells.
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.


METHODS
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.
Hayashi
Tsukasa
T
KAINOS Laboratories Inc., 2-38-18 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Kobayashi
Hiroyuki
H
Miyachi
Hayato
H
Ohshima
Toshio
T
Ujiiye
Takeshi
T
Kawase
Masako
M
Hotta
Tomomitsu
T
Takemura
Yuzuru
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007938:Leukemia / Q000097:blood / Q000235:genetics*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000097:blood / Q000235:genetics; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000378:metabolism; D009190:Myelodysplastic Syndromes / Q000097:blood / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021161:Self-Sustained Sequence Replication / Q000379:methods; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D012333:RNA, Messenger; D011990:Receptors, Transferrin