Samples from patients with chronic myelogenous leukemia

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					                                    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.



                                  Keywords:
                                  tumor cell detection, RT-PCR, mRNA Isolation
                                                                                                         I ntroduction

                                  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
                                  A
PCR




                                       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
                                                                                                                                                      CONTENTS
R   esults and Discussion
                                                        References
For the validation of the mRNA Isolation Kit for        [1] Rowley, J.D. (1993) Nature 243: 290-293.
Tissue and to determine the sensitivity of the assay,   [2] Melo, J.V. (1996) Leukemia 10: 751-756.
we added different amounts of t(9;22)-positive total    [3] 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-
                                                        [4] 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).
                                                                                                 tissue

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
not shown).




ROCHE MOLECULAR BIOCHEMICALS                                                                      BIOCHEMICA · No. 1        1999   13
CONTENTS