The recurrent chromosome translocations in leukemia are excellent targets for the tracking of residual disease in patients undergoing treatment. The fusion genes that result from the chromosome rearrangements are specific (in the main) for leukemic cells. PCR assays that use primers complimentary to the fusion partners can be highly sensitive due to very low or absent background. However, some leukemia specific fusion transcripts, such as BCRABL, are found in a significant proportion of healthy individuals (Bose et al., 1998). Sensitivities equivalent to one positive cell in one hundred thousand negative cells can be achieved by nested qualitative RT-PCR reactions. RT-PCR is applicable to the detection of fusion transcripts due to the great distances spanned by the introns in the partner genes. The use of the spliced RNA transcript not only overcomes the problem of long PCR; it increases the sensitivity due to the presence of multiple copies of the fusion transcript rather than the single copy of the rearranged gene. However, the labile nature of RNA is a major disadvantage in the use of RT-PCR assays as diagnostic tests. Not only does RNA degrade as cells lose viability; purified RNA is vulnerable to the ubiquitous presence of ribonucleases in the laboratory environment. The problems associated with the use of RNA can be minimized with a few sensible precautions in the laboratory and the optimization of specimen collection and delivery.
As quantitative gene expression has been described in great detail in Chapter 7, another complete protocol is not presented. A brief outline of our protocol for the quantitation of BCRABL in patients with CML treated with imatinib is given as an example.
• We perform gene expression TaqMan® assays using 10 pl assays per well. This is possible as 1 pl of cDNA may be dispensed accurately (however it is difficult to dispense such small volumes of concentrated genomic DNA)
• Do not dilute cDNA from patients on imatinib therapy
• Prepare large stocks of diluted probe and primers and store in small aliquots in amber tubes at -20°C
Constituent Volume Volume per per well 96-well plate
Molecular biology grade H2O 3 pl 315 pl
10 pM forward primer 0.3 pl 31.5 pl
10 pM reverse primer 0.3 pl 31.5 pl
• Each well should contain:
2X Master mix 5 pl
Probe and primer mix 4 pl
Template (cDNA or plasmid) 1 pl
• Standard curves for both BCRABL and ABL are used on each run. We use an in-house plasmid construct that contains a full-length b3a2 BCRABL transcript, permitting the same plasmid to be used for both. We use the plasmid standards from Ipsogen to calibrate our own preparations. However, both BCRABL and ABL standards must be purchased, as they are different constructs
• Use triplicate reactions for all tests and controls
• Carry out 50 cycles of PCR to ensure that samples with high Ct values represent true amplification
• Analyze results and examine replicates
• Export results to Microsoft Excel
• Express results as BCRABL/total ABL x 100
• Express individual specimen sensitivity as last reproducible point on standard curve/ABL copy number
Was this article helpful?