Detection of BCR-ABL Chimeric mRNA in Peripheral
Blood and Bone Marrow
Samples from patients with chronic myelogenous
leukemia by RT-PCR in combination with the mRNA
Isolation Kit for Tissue
F. Grünebach, L. Kanz and W. Brugger
Department Med II, Hematology, Oncology, Immunology and Rheumatology, University of Tübingen,
F. Grünebach Otfried-Müller-Straße 10; D-72076 Tübingen, Germany.
tumor cell detection, RT-PCR, mRNA Isolation
Kit for Tissue More than 95% of patients with chronic myelogenous
leukemia (CML) are characterized cytogenetically by
a t(9;22)(q34;q11) reciprocal translocation that results
bstract in transposition of the ABL proto-oncogene from
chromosome 9q34 to 22q11, where it is fused with part
The new Roche Molecular Biochemicals’ mRNA Isola- of the BCR gene (1). Because the t(9;22) translocation
tion Kit for Tissue was tested to prove its usefulness is present in the early chronic phase, the union of the
in life science research studies for the RT-PCR detec- BCR and ABL genes is probably involved in the initi-
tion of t(9;22)-positive tumor cells in peripheral blood ation of the leukemic process (2, 3).
or bone marrow of patients with chronic myelogenous
leukemia (CML). To elucidate the sensitivity of the Cytogenetic analysis-based detection of persistent
assay, t(9;22)-negative cells were spiked with decreas- t(9;22)-positive cells after bone marrow transplanta-
ing amounts of t(9;22)-positive cell line total RNA. In tion is not possible in the majority of patients who are
addition, different patient samples were examined for in hematologic remission (4). Instead, the BCR-ABL
the chimeric mRNA. gene can be efficiently identified by reverse-tran-
scription polymerase chain reaction (RT-PCR) ampli-
fication of its fusion transcript. The chimeric mRNA
includes the B2 or B3 exon from the M-BCR region of
BCR and the A2 exon from ABL to form the so-called
B2A2 or B3A2 fusion mRNAs respectively.
In preparation for the RT-PCR, mRNA was isolated as
described in the package insert of the mRNA Isolation
Kit for Tissue: briefly, the Iysate was diluted with
hybridization buffer containing biotin-labeled
oligo(dT), and precipitates were removed by centrifu-
gation. Then, streptavidin-magnetic particles were
added to the supernatant, and the tubes were trans-
Figure 1: RT-PCR detection of t(9;22)-positive tumor cells in peripheral blood or bone ferred to a magnetic stand. Repeated steps of separa-
marrow samples using the mRNA Isolation Kit for Tissue. Lysates of 10 6 HL-60 cells were tion and resuspension washed the mRNA that was
spiked with decreasing amounts of K-562 total RNA (3.0 ng to 3.0 pg). Subsequently, either total immobilized onto streptavidin-magnetic particles.
RNA or poly(A) + mRNA was isolated and submitted to ß 2-microglobulin (B2M) and BCR-ABL Finally the pure mRNA was eluted from the particles
RT-PCR amplification as described. Additionally, three CML patient samples were analyzed with 11 µl water.
(PB: peripheral blood; BM: bone marrow) by RT-PCR. Patient #1 shows the shorter 320 bp frag-
ment corresponding to the B2A2 fusion transcript. The PCR products were resolved on an 3%
Agarose gel. Vlll: Roche Molecular Biochemicals’ Molecular Weight Marker Vlll.
12 BIOCHEMICA · No. 1 1999 ROCHE MOLECULAR BIOCHEMICALS
R esults and Discussion
For the validation of the mRNA Isolation Kit for  Rowley, J.D. (1993) Nature 243: 290-293.
Tissue and to determine the sensitivity of the assay,  Melo, J.V. (1996) Leukemia 10: 751-756.
we added different amounts of t(9;22)-positive total  Gale, R.P., Grosveld, G., Canaani, E., and Goldman J.M.
(1993) Leukemia 7: 653-658.
RNA from the leukemia cell line K-562 to 106 nega-
 Martinelli, G., Sessarego, M., Gasparini, P., Ambrosetti, A.,
tive HL-60 cells. The mRNA isolation protocol in
Frassoni, F., Chiamenti, A.M., and Pignatti, P.F. (1991)
combination with the nested RT-PCR assay was able Bone Marrow Transplant. 7 Supplement 2: 21.
to detect approximately one positive cell (30 pg of
total RNA) in a background of 106 negative cells.
When using concentrated total RNA for RT-PCR
amplification, 0.3 ng of K-562 RNA could be detected Product Cat. No. Pack Size
(Figure 1). The high quality of the different mRNA mRNA Isolation Kit 1 978 608 25(100; 250) isola-
and total RNA samples was assessed by the RT-PCR for Tissue ‡ tions from 100 mg
(25 mg; 10 mg) of
amplification of β 2-microglobulin mRNA (Figure 1).
In all samples, only the expected PCR fragment for
cDNA amplification was obtained. In addition, the
contamination of the mRNA samples with genomic For the complete version of this article, please visit
DNA was ruled out by the amplification of mRNA our web site at
without cDNA synthesis. No sample gave a positive http://biochem.roche.com/Prod_inf/Biochemi/
result (data not shown). No1_99/B199grue.htm
In our RT-PCR assay, the use of poly(A) mRNA
instead of total RNA resulted in a factor 10 higher
sensitivity. Additionally, when only small sample
sizes were available (10 4 to 105 cells), the mRNA
Isolation Kit for Tissue could still be applied (data
ROCHE MOLECULAR BIOCHEMICALS BIOCHEMICA · No. 1 1999 13