Detection and direct sequence identification of BCR-ABL mRNA in Ph+ chronic myeloid leukemia.
The reverse transcriptase-polymerase chain reaction (RT-PCR) for BCR-ABL mRNA is increasingly used to diagnose and monitor patients with Ph+ chronic myeloid leukemia (CML). We investigated an alternative approach to detect BCR-ABL mRNA in CML in order to overcome some of the potential drawbacks of RT-PCR. Nucleic acid sequence based amplification (NASBA) is a homogeneous, isothermal, in vitro process that provides the direct amplification of RNA. Peripheral blood from seven patients with Ph+ CML and Ph+ EM-2 cells were investigated by NASBA and RT-PCR. A nested set of four primers flanking the BCR-ABL junction was used in two serial NASBA reactions performed for 2 hours. The two methods were fully concordant for detection of transcripts with bcr3-abl2 and bcr2-abl2 junctions. Ethidium bromide fluorescence with NASBA indicated in repeated experiments that similar quantities of total RNA from patient material contained different amounts of BCR-ABL mRNA. The data suggest that direct amplification of RNA is suitable for identifying and monitoring patients with Ph+ CML and may provide a means to quantify BCR-ABL mRNA levels.
Sooknanan
R
R
Cangene Corporation, Mississauga, Ontario, Toronto, Canada.
Malek
L
L
Wang
X H
XH
Siebert
T
T
Keating
A
A
D001483:Base Sequence; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis / Q000235:genetics*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000097:blood* / Q000737:chemistry; D017423:Sequence Analysis, RNA
D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl