Overexpression of CDK6. (A) Quantitative real-time PCR was performed using the LightCycler system with FastStart DNA Master SYBR Green I kit according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Primers from the CDK6 gene are as follows: forward (5′-AGAAGAAGACTGGCCTAGAG) and reverse (5′-TGGAAGTATGGGTGAGACAGG). Primers from the β-2-microglobulin (β2-MG) gene used to evaluate correcting target molecule amounts are as follows: forward (5′-CCAGCAGAGAATGGAAAGTC) and reverse (5′-GATGCTGCTTACATGTCTCG). Amplification of specific transcripts was confirmed by a melting curve analysis. For PCR calibration, a serial 10-fold dilution series for each gene (ranging from 10⁶ to 10 copies) was amplified and the assay was found to be linear over at least 5 orders of magnitude (CDK6 slope, –3.47; β2-MG slope, –3.43). The absolute copy amount of each sample was obtained by the mean of the following ratio: ([copy number of the CDK6 gene]/[copy number of β2-MG]) × 100. (B) Total cellular proteins were analyzed by Western blotting using anti-CDK6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The same blot was probed for actin to control for equal protein loading. In all samples, tumor cells represented more than 80% of the nucleated cells. RNA and protein were extracted from bone marrow or blood samples from patients with the CDK6 gene rearrangement (FIO, DUP, LAN), from patients with B-CLL without the CDK6 gene rearrangement (B-CLL1 to B-CLL4), and from normal mononuclear peripheral blood cells (C1 to C5).