Defective p66Shc expression in CLL B cells correlates with an alteration in the balance of antiapoptotic and proapoptotic Bcl-2 family members. Quantification by real-time RT-PCR of the levels of Bcl-2 and Bcl-xL mRNA (A) or Bax and Bak (B) in PBLs from the 64 patients with CLL, grouped in the M-CLL and U-CLL subsets. Transcript levels were normalized to the expression level of GAPDH. For each patient, Syber green runs were performed on duplicate samples of cDNAs from 2 independent reverse transcription reactions, each carried out on 400 ng of total RNA. RNA from the same healthy donor (C1) was included in duplicate in each run as reference to normalize the data obtained in the individual experiments. The ΔΔC_T method was applied as a comparative method of quantification, using as a reference the average of all the Ct data obtained on healthy donors. No significant variation among healthy donors was observed (see supplemental Figure 3). The histograms summarize all the data obtained on the control and CLL subsets. ***P ≤ .001, **P ≤ .01. Error bars indicate SD.