Soon Pal Suh, Seung Jung Kee, Woo Hyun Lim, Jeong Won Song, Sang Khoo Lee, Jong Phil Kim, Jong Hee Shin, Dong Wook Ryang
Multiplex In-cell Reverse Transcription-Polymerase Chain Reaction for the Simultaneous Detection of p210 and p190 BCR-ABL mRNAs in Chronic Myeloid Leukemia and Philadelphia-Positive Acute Lymphoblastic Leukemia Cell Lines
We designed a novel multiplex in-cell reverse transcription-
polymerase chain reaction method for the simultaneous
detection and differentiation of p190 and
p210 BCR-ABL mRNAs within single cells from the human
chronic myeloid leukemia and Philadelphia positive
acute lymphoblastic leukemia. Human K562
chronic myeloid leukemia and SUP B-15 Ph+ acute
lymphoblastic leukemia cell lines were used as positive
controls for p210 and p190 BCR-ABL mRNAs, respectively.
HL60 cell line was used as a negative control.
After the leukemia cells were fixed and
permeabilized, without extracting nucleic acids, the
mRNAs were reverse transcribed to cDNAs, and the
cDNAs were amplified by multiplex polymerase chain
reaction with fluorescent primers specific for p190 and
p210 BCR-ABL mRNAs. After transfer onto glass slides
by cytospin, the amplified cells were detected by fluorescence
microscopy. Fluorescence microscopy after
propidium iodide or 4?,6-diamidino-2-phenylindone
counterstaining showed that the positive K562 cells
exhibited a yellow-green fluorescent cytoplasm
around a red nucleus, and that the positive SUP B-15
cells exhibited an orange cytoplasm around a blue nucleus.
Only the red or blue nucleus was visible in respective
negative HL60 cells. The specificity of amplification
was confirmed by the absence of a signal when
control experiments were performed either with
RNase digestion of mRNA or without reverse transcriptase/Taq polymerase. We conclude that the multiplex
in-cell reverse transcription-polymerase chain
reaction method is capable of simultaneously detecting
and differentiating the p210 and p190 BCR-ABL
mRNAs of chronic myeloid leukemia and Philadelphia-positive
acute lymphoblastic leukemia cells, and that it
may be useful in quantitatively monitoring the minimal
residual disease during therapy.
Clinical Chemical Laboratory Medicine, Walter de Gruyter
Print ISSN: 1434-6621
Volume: 38, 09/2000
Pages: 939 - 944
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