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These data demonstrate for this case that the ΔCt found to be outside the normality range is not due to primer/probe mismatch but may be the result of a partial LOI of the maternal allele.
Of the 14 GBMs with Ct ratio outside the normality range only five among methylated cases had MGMT methylation and the ΔCt of SNURF had some impact on the final calculation for the m MGMT/u MGMT ratio in only one of 70 cases (1.42%, sample BF215).
The SNURF promoter of the 14 cases with Ct ratio [(m SNURF - u SNURF)] outside the normality range was sequenced to verify whether they showed the simultaneous presence of both methylated and unmethylated cytosine in each of the CpGs interrogated by the primers and beacon probes.
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A sample of 24 healthy controls was used to estimate the normality range of the differences between methylated and unmethylated SNURF alleles; the normality range was estimated as the mean value ± 1.96 * standard deviation.
For BMD, it was 5.54, falling within the normality range for n = 74 of 4 to 5.87.
Values outside the SNURF normality range were detected in 14 out of 159 GBM (8.8%; see the Additional file 1 for details).
Hematological values outside the normal range were infrequent.
Biochemistry values outside the normal range were also infrequent.
No creatinine values were outside the normal ranges.
The q value for PTH was 6.38, falling outside the 4.17 to 6.07 normality range for n = 95.
Regarding LH, 71% of the individuals studied had serum levels within the WHO (1999) normality range (2.5 9.8 IU/L).
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