GSTT1 and GSTM1 gene copy number analysis in paraffin-embedded tissue using quantitative real-time PCR.
GSTT1 and GSTM1 genes possess an inherited deletion associated with a lack of enzyme activity. The heterozygous condition of this deletion is difficult to determine in low-quality DNA with existing PCR protocols. We designed and validated a multiplex real-time PCR assay by adapting the DeltaDeltaCt relative quantification method for the analysis of GSTT1 and GSTM1 markers to accurately differentiate the three genotypes ( *1/1, *1/0, and *0/0) in degraded DNA from formalin-fixed paraffin-embedded tissue. Gene copy number values obtained provide for unambiguous homozygous and heterozygous differentiation. The efficacy shown by the PCR assay endorses its usefulness for complete genotyping of glutathione S-transferases in archival tissues.
Bediaga
Naiara G
NG
Servicio de Genómica, Banco de ADN, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria, Spain.
Alfonso-Sánchez
Miguel A
MA
de Renobales
Mertxe
M
Rocandio
Ana M
AM
Arroyo
Marta
M
de Pancorbo
Marian M
MM
D018628:Gene Dosage; D005838:Genotype; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D016612:Paraffin Embedding; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D005982:Glutathione Transferase; C413545:glutathione S-transferase T1