Quantitative Real-Time PCR for BRCA1/2 Expression

Primers and probes for the TATA box-binding protein (TBP;
endogenous RNA-control) were used as previously
described.^{13 (link)} Primers and probes for BRCA2 [GenBank: NM_000059.3] were determined with the assistance of
the computer program Primer Express (Life Technologies, Carlsbad, CA, USA).
BRCA2 forward primer: 5′-GAA AAT CAA GAA AAA
TCC TTA AAG GCT-3′; BRCA2 reverse-primer:
5′-GTA ATC GGC TCT AAA GAA ACA TGA TG-3′; BRCA2TaqMan probe: 5′-FAM-AGC ACT CCA GAT GGC ACA ATA AAA GAT CGA AG-3′-TAMRA. Primers
and probe for BRCA1 were purchased from Applied
Biosystems (Foster City, CA, USA, Applied Biosystems Assay ID: Hs01556193_m1). PCR
reactions were performed as previously described.^{13 (link)} Each experiment included a
standard curve with five cDNA concentrations, a positive control sample (OVCAR-3
carcinoma cell-line), 40 patient samples and a no template control. The standard
curves were generated using serially diluted solutions of standard cDNA derived
from the HTB-77 carcinoma cell line. The target mRNA quantity in each sample was
determined from the relative standard curve, data normalization was carried out
against TBP, the endogenous RNA-control and expressed in arbitrary units
corresponding to the dilution factors of the standard RNA preparation. Real-time
PCR assays were conducted in duplicates for each sample, and the mean value was
used for calculation.