TABLE 2.
Quantitative analysis of p53 gene loss in TgT121p53+/+ tumorsa
| Sample no. | Tissue | Mouse genotype | 2−ΔΔCtb | No. of assays | No. of wild-type p53 alleles |
|---|---|---|---|---|---|
| 1 | Tail | p53+/+ | 1.0 | 4 | 2 |
| 2 | Tail | p53+/− | 0.33 ± 0.24 | 4 | 1 |
| 3 | Tail | p53+/− | 0.21 ± 0.02 | 2 | 1 |
| 4 | Tumor (I) | TgT121p53+/− | 0.17 ± 0.05 | 4 | 1c |
| 5 | Tumor (I) | TgT121p53+/− | 0.11 ± 0.04 | 2 | 0 |
| 6 | Tumor (I) | TgT121p53+/+ | 0.17 ± 0.01 | 2 | 1d |
| 7 | Tumor (I) | TgT121p53+/+ | 0.48 ± 0.14 | 2 | 1 |
| 8 | Tumor (I) | TgT121p53+/+ | 0.14 ± 0.01 | 2 | 0d |
| 9 | Tumor (II) | TgT121p53+/+ | 0.73 ± 0.02 | 2 | 2 |
| 10 | Tumor (II) | TgT121p53+/+ | 1.21 ± 0.62 | 2 | 2 |
| 11 | Tumor (II) | TgT121p53+/+ | 1.56 ± 0.69 | 3 | 2 |
Real-time PCR was performed to determine the status of the wild-type p53 alleles in TgT121p53+/+ terminal tumors (see Materials and Methods). Among six TgT121p53+/+ tumors, all three type I tumors showed loss of at least one wild-type p53 allele while all three type II tumors retained both wild-type alleles. Two TgT121p53+/− tumors shown to have lost the wild-type p53 allele by allele-specific PCR and CGH were used as controls.
ΔΔCt = [sample Ct(p53) − sample Ct(β-actin)] − [p53+/+ control Ct(p53) − p53+/+ control Ct(β-actin)], where Ct is the number of cycles required to reach a threshold based on linear amplification (see Materials and Methods). Analysis of standard samples indicated that copy numbers of 2, 1, and 0 are indicated by 2−ΔΔCt values of >0.6, between 0.15 and 0.6, and <0.15, respectively.
Although the value obtained here indicates detection of a wild-type allele in this TgT121p53+/− tumor, this is likely due to the contamination of nontumor tissue in the dissection procedure. Previous conventional PCR analysis and CGH all showed that tumor 4 had p53 LOH.
The 2−ΔΔCt value for tumors 6 and 8 that arose in mice with two wild-type alleles is near the low end of the single-copy range. Further analysis will be required to determine with certainty whether one or both wild-type alleles are absent in these tumors. However, there is clearly loss of at least one allele.