real-time-quantification 09.05.2000 19:18 Uhr Seite 8 Real-time Quantification of BCR-ABL mRNA Transcripts Using the LightCycler-t(9;22) Quantification Kit Heiko Wittor 1, Hermann Leying 1, Andreas Hochhaus 2, and Rob van Miltenburg 1 1 Roche Molecular Biochemicals, Penzberg, Germany 2 III. Medizinische Universitätsklinik, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany ly different starting concentrations, so that the initial Introduction target concentration can be determined in a single PCR. The concentration of BCR-ABL transcripts is determi- Literature indicates that in 95 % of all subjects with ned relative to the number of transcripts of a control chronic myeloid leukemia (CML) and in 25-30 % of gene, glucose-6-phosphate dehydrogenase (G6PDH). subjects with acute lymphoblastic leukemia (ALL) a The entire procedure from sample preparation to reciprocal translocation between the long arms of quantitative result is performed in 4.5 hours. chromosome 9 and chromosome 22 [t(9;22)] can be found. This translocation or the resulting fusion pro- duct can be detected by a number of techniques, CLER including fluorescent in situ hybridization, Southern CY blotting, western blotting and reverse transcriptase TThe LightCycler System polymerase chain reaction (RT-PCR). Of these techni- The LightCycler System is based on the amplification LIGHT ques, RT-PCR for the chimeric fusion transcript BCR- of target sequences using alternating heated and ABL has received most attention in relation to the ambient temperature cycles. Samples are contained in detection of minimal residual disease because of its glass capillaries with high surface-to-volume ratio, high sensitivity (1). Attempts to research the remission allowing a rapid heat exchange between the air in the and relapse events, occurring in the body of persons thermal chamber and the reaction components. By with CML after treatment with interferon-a or after combining air as a medium for thermal cycling with bone marrow transplantation, requires sensitive tech- capillaries as reaction vessels, the LightCycler niques that detect smallest amounts of residual leuke- Instrument enables rapid PCR by reducing the required mic cells. Research concerning a possible correlation cycle times. Typically 40 cycles can be performed in between a positive nested RT-PCR signal and the pro- just 20 – 30 minutes. gression of the leukemia is hampered by the non- quantitative nature of conventional nested RT-PCR. The LightCycler Instrument's optical unit continuously Quantitative RT-PCR approaches that allow quantifica- monitors the formation of PCR product by measuring tion of the transcript by competitive RT-PCR have been the amount of Hybridization Probes annealing to the developed (2,3). The technique is based on serial dilu- target sequence in every cycle. Hybridization probes tions of competitor contructs added to a constant emit a fluorescent signal, that directly correlates to the amount of sample cDNA. Quantification is achieved by concentration of target. Comprehensive information on comparison of the relative band intensities of target the LightCycler System and the various fluorescent for- and competitor PCR product on agarose gel. However, mats that can be used to monitor PCR, can be found on this method is very laborious since several concentra- tions of the competitor have to be used to cover the expected concentration range. http://biochem.roche.com/lightcycler In this article we present the LightCycler-t(9;22) Quantification Kit which is a research tool designed for or can be requested from your Roche Molecular the further study of possible relationships between the Biochemicals' representatives. level of BCR-ABL fusion transcripts expression, stage of the disease, and response to treatment. The LightCycler System approach for the quantitative detection of the BCR-ABL fusion transcripts is based on real-time detection. The wide dynamic range of real-time PCR enables simultaneous analysis of samples with high- 8 BIOCHEMICA · No. 2 m 2000 ROCHE MOLECULAR BIOCHEMICALS real-time-quantification 09.05.2000 19:18 Uhr Seite 9 valent to approximately 3.3 ml whole blood) was rever- Experimental Design se transcribed using random primers in a single 20 µl reaction. Five microliters of the cDNA was used for Primers and probes each of the specific PCRs (Figure 2). All reagents The BCR-ABL fusion transcript can be formed by diffe- required for the cDNA synthesis and for the PCR step rent recombinations of the BCR-gene from chromoso- are provided in the kit. me 22 and the ABL-gene from chromosome 9. Most breakpoints are mapped to the major (M-bcr) and 1. RT-buffer minor (m-bcr) breakpoint cluster regions. Three fusion 2. Random Primer p(dN)6 transcripts (b2a2, b3a2 [M-bcr] and e1a2 [m-bcr]) 3. Deoxynucleotide Mix represent about 96 % of the described BCR-ABL 4. AMV Reverse Transcriptase breakpoints in CML. 5. RNase Inhibitor 6. Reaction Mix, 10x To detect the most relevant fusion transcripts a PCR contains Taq DNA Polymerase, reaction buffer, and dNTP mix (with dUTP instead of dTTP) was designed with 3 amplification primers; one for- 7. t(9;22) Detection Mix, 10x conc. ward primer in BCR exon e1 and a second forward pri- contains ready-to-use primer and Hybridization Probe mix mer in BCR exon b2. Either primer combine with the specific for t(9;22) fusion gene reverse primer in ABL exon a4 (Figure 1). All products 8. G6PDH Detection Mix, 10x conc. that are amplified by this set of primers contain the abl contains ready-to-use primer and Hybridization Probe mix exon a3. Therefore, Hybridization Probes were de- specific for the human glucose-6-phosphate dehydrogen- signed for this region. Note that this primer/probe ase (G6PDH) gene 9. G6PDH RNA Standards, 3 concentrations CLER combination also enables the detection of the rare containing in vitro transcribed G6PDH RNA b2a3 and b3a3 fusion transcripts . CY 10. t(9;22) Positive Control containing mRNA isolated from K562 cells As a control for the amount, the integrity and the qua- LIGHT 11. H2O, sterile, PCR Grade lity of the mRNA sample a control gene (G6PDH) with an expression level similar to the BCR-ABL transcripts Ý Table 1: Contents of the LightCycler t(9;22) Quantification Kit. was selected. Amplification primers and Hybridization Probes for the G6PDH gene were selected for highly comparable amplification efficiency when compared with the BCR-ABL PCR. Known concentrations of in vitro transcribed RNA of the G6PDH gene are used for the generation of a standard curve. Relative concentra- tions of BCR-ABL mRNA and G6PDH mRNA from the samples are then determined by comparison to this standard curve. Ý Figure 1: Location of amplification primers and Hybridi- zation Probes for detection of b2a2, b3a2, and e1a2 transcripts. Samples mRNA was isolated from up to 5 ml of heparinized blood samples, using the mRNA Isolation Kit for White Blood Cells from Roche Molecular Biochemicals. In principle, total RNA can also be used. Two-step RT-PCR Û Figure 2: cDNA was prepared from the purified mRNA using the Flow diagram of two- reverse transcriptase components provided in the step RT-PCR for analysis LightCycler-t(9;22) Quantification Kit (see Table 1 for of BCR-ABL mRNA kit contents). Two-thirds of the isolated mRNA (equi- and G6PDH mRNA. ROCHE MOLECULAR BIOCHEMICALS BIOCHEMICA · No. 2 m 2000 9 real-time-quantification 09.05.2000 19:19 Uhr Seite 10 Cycling After an initial denaturation step of 30s at 95°C, ampli- fication was performed in a three-step cycle procedu- re (denaturation 95°C, 1s, ramp rate 20°C/s; annealing 64°C, 10s, ramp rate 20°C/s; and extension 72°C, 26s, ramp rate 2°C/s) for 45 cycles. A RResults Results are shown in Figures 3 and 4. For clarity, the amplification curves for the different capillaries in the experiment are shown in different screens. Figure 3 displays the fluorescent signal of the G6PDH standards and the standard curve. This standard curve was con- structed by plotting the concentration of the standard versus the cycle number at which the fluorescent sig- nal could first be differentiated from the background using the second derivative maximum algorithm in the B CLER LightCycler Software. The G6PDH concentrations of CY the samples (Figure 4a) and the BCR-ABL concen- trations (Figure 4b) were calculated by the LightCycler Ý Figure 4: Amplification plots for five blood samples. LIGHT Software, using this standard curve. A. Detection of the G6PDH target. Note that the concentrations are The final step is the calculation of the ratio BCR- comparable for samples 2-5 whereas the G6PDH mRNA in ABL/G6PDH (Table 2). sample 1 was detected several cycles later. This is indicative of poor quality sample material (e.g. RNA degradation). B. Detection of the BCR-ABL fusion transcript. Concentrations were different for all five samples. No signal was detected in sample 1, possibly due to poor quality of the sample. A repeat of the analysis with fresh material would be recommended. Sample Calculated concentrations in pg BCR-ABL G6PDH BCR-ABL/G6PDH Standard 2 ng – 2070 – Standard 2 pg – 1.6 – Standard 0.6 pg – 0.69 – Blood sample 1 n.d. 0.438 – Blood sample 2 6.95 72.7 0.096 Blood sample 3 37.5 49.8 0.753 Blood sample 4 2.02 28.4 0.071 Blood sample 5 205 66.7 3.07 A Ý Table 2: Calculation of the BCR-ABL/G6PDH ratio for the blood samples. The higher the BCR-ABL/G6PDH ratio is, the more leuke- mic cells were present in the samples analyzed. Û Figure 3: Amplification curves for the G6PDH standards. A. Fluorescence signal development during PCR. Standards were 2 ng (duplicates), 2 pg (duplicates) and 0.6 pg (triplicates).