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RT-PCR Primers for the Study of Apoptosis

O. Mineral oil (100 l) was added on each tube.

The PCR amplifications were cycled using the RoboCycler temperature cycler and the following parameters: 1 cycle of 94C for 5 minutes; 40 cycles of 94C for 1 minute, 60C for 2 minutes and 72C for 2 minutes; 1 cycle of 72C for 10 minutes. The reactions were stored at 6C. From each PCR amplification, 20 l was analyzed by electrophoresis on a 1.2% agarose gel in TAE buffer with 1 mg/ml of ethidium bromide for staining. The results were visualized using Stratagenes Eagle Eye II still video system.

Mouse ICE Family

Figure 1

A mouse macrophage cDNA library was used as template for the PCR amplifications with two sets of primers (Figure 1). One set, mouse ICE (a,b,g), amplifies the ICE gene and distinguishes the splice variants of ICE. These variants generate PCR products of different lengths: 610-bp PCR product for ICE-a, 520-bp PCR product for ICE-b, and 380-bp PCR product for ICE-g. In contrast, the ICE (all) primer set does not distinguish among the splice variants and generates a 470-bp product for all ICE variants.

Human ICE Family and Fas Antigen

Figure 2

To test human ICE-b , CPP32, ICE-Lap-3, and Fas antigen RT-PCR primers, five human cDNA libraries were used as templates (Figure 2 and Figure 3). Human genomic DNA was used as the genomic DNA control, and mouse and rat cDNA were tested as negative controls. Each amplification yielded
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