Fig. 1

Comparison of bcl-2 relative expression ratio (RER) in peripheral blood CD4+ T cells among healthy volunteers, asbestosis patients and malignant mesothelioma (MM) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood of healthy donors and from patients with asbestosis and MM using a Ficoll–Hypaque density gradient (Separate-L; Muto Pure Chemicals, Tokyo, Japan). For the isolation of CD4+ T cells, PBMCs were further separated using magnetic cell separation (MACS) CD4 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The enriched cells were >90% pure as determined by flow cytometry. Specimens were taken from healthy volunteers and patients from whom informed consent had been obtained. The Institutional Ethics Committee of Kawasaki Medical School, Hyogo College of Medicine, and Okayama Rosai Hospital approved the project. A fluorescence thermocycler (Mx3000P QPCR System; Stratagene, La Jolla, CA) was used for real-time reverse transcriptase (RT)-PCR experiments following the instructions of the manufacturer. With this technique, the fluorescence-labeled amplification product is measured continuously. Total RNA obtained from CD4+ T cells isolated from peripheral CD4+ T cells was extracted using an RNA Bee kit (Tel-Test, Friendswood, TX), and 5 μg of RNA was reverse-transcribed with standard methods using a RevertAid H Minus First Strand cDNA Synthesis lit (Fermentas, Ontario, Canada). An amount of cDNA equivalent to 50 ng of RNA served as the template for PCR in a volume of 20 μl (each primer and SYBER Premix Ex Taq; TaKaRa, Japan). The primers for bcl-2 and gapdh were added to the same reaction tube at the optimal concentration for each primer set, and PCR was performed. The primers were as follows: Bcl-2 [5′-TGATGTGAGTCTGGGCTGAG-3′ (forward: Fw) and 5′-GAACGCTTTGTCCAGAGGAG-3′ (reverse, Rv)]; Bax [5′-AGTAACATGGAGCTGCAGAGG-3′ (Fw) and 5′-ATGGTTCTGATCAGTTCCGG-3′ (Rv)]; GAPDH [5′-GAGTCAACGGATTTGGTCGT-3′ (Fw) and 5′-TTGATTTTGGAGGGATCTCG-3′ (Rv)]. The relative expression of various target genes, such as bcl-2, was calculated when real-time RT-PCR was performed. The relative level of the target gene is expressed as ½[B − A], with gapdh expression being 1.0, and A = number of PCR cycles required to reach a certain intensity of fluorescence for the gapdh product, and B = number of PCR cycles required to reach the same fluorescent intensity for the target gene product (bcl-2) derived from the same sample. PCR products were confirmed to be successfully amplified by standard agarose gel electrophoresis and staining with ethidium bromide. Comparisons of the results for relative gene expression and proliferation assayed by real-time RT-PCR were analyzed using Fisher’s parametric least significant difference (PLSD) test