The method used to assess Cby1 transcript levels is a semi-quantitative one intended to evaluate the differences in Cby1 transcript or protein levels from samples of chronic myeloid leukemia patients and healthy persons (pooled to avoid individual differences and used for all quantifications).
Accordingly, the PCR signal intensities obtained from the pool of healthy persons was used as reference value (= 1) and those obtained from single patients derived from the densitometric analyses. In other terms, we never assumed to take the band intensity as a measure of Cby1 transcript amount. We rather considered that the differences in PCR band intensities would reflect differences in Cby1 transcript amounts in chronic myeloid leukemia and normal cells. As a matter of fact, we are perfectly aware that PCR band intensity can not be taken as a measure of transcript amount. But the comparison between PCR band intensities from different samples and/or under different conditions is widely used and commonly accepted to appreciate the quantitative differences in given transcript(s).
Just to dissipate any doubt about our technical accuracy we want point out that PCR amplification reactions reported in our paper were simultaneously carried on RT products of either mononuclear cell fractions or CD34+ cell from CML patients and pooled healthy persons. Signal intensities of PCR bands were then measured using a dedicated software (IMAGEJ 1.44p Launcher software from National Institutes of Health, Bethesda, MD, USA), which considered the differences among patients and normal controls relative to beta 2 microglobulin as control for amplification. Similar results have been obtained in three separate experiments for each sample, supporting the congruity of our method.
Indeed, we performed experiments using real time PCR. Such experiments confirmed the results obtained with the above mentioned semi-quantitative PCR. However, they were not included in our paper for the lack of proper reference gene(s), in particular, in a cell compartment, the leukemic stem cell compartment, whose genetic profile is still elusive. The person who made the comment refers to the “state of art” in the ’80s. He should therefore know how long and contended was the discussion about the gene to keep as reference for BCR-ABL1 transcript evaluation for monitoring the outcomes of chronic myeloid leukemia therapy with imatinib.
With the hope that our answer has cleared any concern about our work, we suggest a smoother mood in the evaluation of other people work (not only us but the journal Editorial office).