![Figure 1. Analysis of CTA expression by RT-PCR, Q-PCR, and antibody staining in bulk populations and in single MM cells. Primer design for all CTAs examined spanned intronic DNA (available on request). Expression of PASD1 was examined by RT-PCR for 30 cycles and compared with known CTAs in MM using identical amounts of cDNA input from RNA (2 μg), reverse transcribed with oligo-dT (A). PASD1 expression was further examined after 40 cycles. Several normal lymphoid tissues were also analyzed, including specific lymphocyte populations not probed previously. In purified B cells, in vitro–generated plasmablasts, plasma cells, T cells, and spleen and bone marrow mononuclear cells (MNCs), we failed to detect PASD1 expression either by high amplification cycle numbers or by Q-PCR (data not shown). Two Burkitt lymphoma cell lines (Ramos, Daudi) were used as negative controls (A). Q-PCR used Taqman primers (PASD1: Hs00542865_m1; β-actin: Hs99999903_m1; Applied Biosystems, Warrington, United Kingdom). Relative expression was calculated against reference sample (Ramos) as 2–[DCt(Sample) – DCt(Reference)], where DCt indicates Ct(PASD1) – Ct(β-actin). Presentation diagnostic samples were MM-(P1-9), pretreated MM-(T10-16); ND indicates not detected (B). Immunoperoxidase labeling showing PASD1 protein expression in testis and in the nuclei of THIEL cells, with JJN3 cells negative (inset), and in the nuclei (arrowed) of tumor cells in 1 of the 2 cases of MM found to be positive (C; RPMI8226 and second MM case staining not shown). Single cells obtained by flow cytometry deposition of 2 MM cell lines expressing high (Jim1) or low (RPMI 8226) levels were analyzed by nested RT-PCR for PASD1 expression, and for each line, upper and lower panels correspond to cell numbers 1 to 24 and 25 to 48, respectively (D). Both splice variants were detected in the 2 lines (± exon 8), yielding 298- and 215-bp amplification products. Each amplification product from bulk populations or single tumor cells was eluted and identity confirmed by DNA sequence analysis. Images were obtained using a Zeiss Axioskop microscope (Zeiss, Welwyn Garden City, United Kingdom) with nonimmersion 10×/0.25 NA (testis, JNN, and MM) and 40×/0.65 (THIEL and MM) Acroplan objectives. Image acquisition was performed using a Micropublisher 5 camera (Q-Imaging, Wokingham, United Kingdom) and Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA). All subsequent processing was performed using Adobe Photoshop 8.0.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/108/12/10.1182_blood-2006-04-014621/4/zh80230604720001.jpeg?Expires=1735593566&Signature=oImycBvXcGsl8wcOMVUvlhRcpRhDvYjwkVb2bqNmmL2JtTBjJHDhOieBfcENNCBwCk8dtahqELLaYNCmlx0vryHhPz-oSa-~u5ZSeuUtuSb7nQdMyIlpRP7T3LcmUWgjuHx5zUTGJFfuy4uKAJydVI12-QMAT6nccHa~Kqbaj-dBo69fCDrvUqkct4CvfKRAZ0OcDgxh70gTD-a3cd6IojbLLcKOdtNT-t8k1YKA4nGbGLzSh1wo9ULgnxh9xs9SzNdkqXZ-rtNIvo9PbkJ0okNYYwZdE~98S2YcC4FrdTCLT0udlvLBybunhYrvYJVN7OA9B9WA4qyOQjbAYaxB7Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Analysis of CTA expression by RT-PCR, Q-PCR, and antibody staining in bulk populations and in single MM cells. Primer design for all CTAs examined spanned intronic DNA (available on request). Expression of PASD1 was examined by RT-PCR for 30 cycles and compared with known CTAs in MM using identical amounts of cDNA input from RNA (2 μg), reverse transcribed with oligo-dT (A). PASD1 expression was further examined after 40 cycles. Several normal lymphoid tissues were also analyzed, including specific lymphocyte populations not probed previously. In purified B cells, in vitro–generated plasmablasts, plasma cells, T cells, and spleen and bone marrow mononuclear cells (MNCs), we failed to detect PASD1 expression either by high amplification cycle numbers or by Q-PCR (data not shown). Two Burkitt lymphoma cell lines (Ramos, Daudi) were used as negative controls (A). Q-PCR used Taqman primers (PASD1: Hs00542865_m1; β-actin: Hs99999903_m1; Applied Biosystems, Warrington, United Kingdom). Relative expression was calculated against reference sample (Ramos) as 2–[DCt(Sample) – DCt(Reference)], where DCt indicates Ct(PASD1) – Ct(β-actin). Presentation diagnostic samples were MM-(P1-9), pretreated MM-(T10-16); ND indicates not detected (B). Immunoperoxidase labeling showing PASD1 protein expression in testis and in the nuclei of THIEL cells, with JJN3 cells negative (inset), and in the nuclei (arrowed) of tumor cells in 1 of the 2 cases of MM found to be positive (C; RPMI8226 and second MM case staining not shown). Single cells obtained by flow cytometry deposition of 2 MM cell lines expressing high (Jim1) or low (RPMI 8226) levels were analyzed by nested RT-PCR for PASD1 expression, and for each line, upper and lower panels correspond to cell numbers 1 to 24 and 25 to 48, respectively (D). Both splice variants were detected in the 2 lines (± exon 8), yielding 298- and 215-bp amplification products. Each amplification product from bulk populations or single tumor cells was eluted and identity confirmed by DNA sequence analysis. Images were obtained using a Zeiss Axioskop microscope (Zeiss, Welwyn Garden City, United Kingdom) with nonimmersion 10×/0.25 NA (testis, JNN, and MM) and 40×/0.65 (THIEL and MM) Acroplan objectives. Image acquisition was performed using a Micropublisher 5 camera (Q-Imaging, Wokingham, United Kingdom) and Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA). All subsequent processing was performed using Adobe Photoshop 8.0.
