Competitive RT-PCR for D-type cyclins. (A) Schematic presentation of primer locations on the D-type cyclin cDNA sequences is shown, where thick lines indicate the coding regions and thin lines represent truncated non-coding regions. Thick arrows indicate primers used in the competitive RT-PCR. The upstream primer (D1S) is shared in amplification of the three D-type cyclin sequences, while the downstream primers, D1AS, D2AS, and D3AS, are specific to their respective D-type cyclin sequences. D1S is derived from the identical region between cyclin D1, D2, and D3 sequences except for one mismatch in the cyclin D2 sequence (B and C) RNA extracted from MEG01s cells was subjected to RT-PCR in the presence (+) and/or absence (−) of the indicated primer with (B) or without (C) [α-³²P]dCTP. Thin arrows denote PCR products corresponding to cyclins D1, D2, and D3, which were visualized by autoradiography (B) or by ethidium bromide staining (upper part in C). PCR products were analyzed by Southern analysis with cDNA probes for the indicated D-type cyclins (middle part in C). The sizes of 100-bp DNA ladders (lane M) are shown on the left.