1:5 dilution? 1:3? 1:10? Or is diluting cDNA for Real-time PCR a waste of time, effort and perfectly good H2O? In this video, Life Technologies Sr. Field Application Specialist Doug Rains examines whether or not qPCR end-users doing gene expression should dilute cDNA prior to running samples. Read More...

Taqman RT-PCR allows us to measure levels of gene expression. The method is reliable, allows establishment of stable standard curves and gene transcript copies numbers in individual samples can be measured very precisely.

An increasing number of laboratories all over the world have applied this technique to measure gene expression in patients in order to monitor particular genes. There are some general pitfals in RT-PCR, which has to be avoid.

In a recent study, was used Quantitative TaqMan-RT-PCR during patient follow-up to examine WT-1 expression patterns in CML and Ph positive ALL patients in comparison with Bcr-Abl expression.

Based on the preliminary results, the WT-1 expression in CML patients generally varies in a pattern similar to that of Bcr-Abl expression. This suggests that WT-1 might provide an alternative parameter for monitoring MRD in patients (either CML or AML).

On the other hand the Bcr/Abl ratio is still the most reliable and sensitive marker for monitoring MRD and guiding the treatment of CML patients. But still, especially in the case of the switch from CML to acute leukemia following a blast crisis, the WT-1 expression contributes useful information about disease progression.

In the cohort of CML-patients, the WT-1/Abl ratio accidentally dropped into the range close to the number in normal blood specimens, but it never reached zero. A possible explanation is that the number of CD34+ cells in bone marrow may be higher than in normal bone marrow. To further explore this possibility, it required to analyze the WT-1 levels durring the whole chimeric period. Some research papers report the usage of WT-1 expression to predict response to STI571 (imatinib mesylate).

At the moment there is no direct evidence from the clinical data to draw conclusions on this issue since the cohort of patients was limited and the used time intervals were quite wide to analyse possible  instantaneous responses.

The results provided some suggestions that the modulation of Bcr-Abl expression possibly precedes the alterations in WT-1 expression. This aligns with the hypothesis that the modulation of tyrosine kinase activity of Bcr-Abl complex can lead to a concomitant change in WT-1 expression.