Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC).
Spontaneous renal cell carcinoma (RCC) occurs with a high frequency in Eker rats carrying a germline alteration of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene. To determine the frequency with which the wild-type allele of the Tsc-2 gene is lost in RCC and the ability of DHPLC to detect loss of heterozygosity (LOH) at this gene locus, fresh-frozen and paraffin-embedded formalin-fixed tumors from heterozygous Eker rats (Tsc-2(Ek/+)) were examined for LOH at the Tsc-2 locus. LOH was determined by quantitation of peak areas of PCR products specific for the mutant and wild-type Tsc-2 alleles. For normal DNA isolated from heterozygous animals, the allele ratio (AR) of mutant to wild-type PCR products was empirically determined to be 1.5+/-0.3 (n=30) and LOH was defined as >2 standard deviations away from this mean, i.e. any AR >2.1. Analysis of 15 spontaneous frozen RCC samples showed LOH in 10/15 samples (66%). Carcinogen-induced tumors exhibited an even higher frequency of LOH, with 6/6 paraffin-embedded, formalin-fixed tumors exhibiting LOH. 100% concordance was observed between the results obtained by DHPLC and traditional methodologies. Therefore, LOH appears to occur with a high frequency in both spontaneous and carcinogen-induced RCC in this animal model and DHPLC is a sensitive and high throughput methodology for detecting this type of genetic alteration.
Kleymenova
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Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Smithville, TX 78957, USA.
Walker
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D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002292:Carcinoma, Renal Cell / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D005615:Freezing; D016147:Genes, Tumor Suppressor; D007680:Kidney Neoplasms / Q000235:genetics*; D019656:Loss of Heterozygosity; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016612:Paraffin Embedding; D051381:Rats; D011922:Rats, Mutant Strains; D012097:Repressor Proteins / Q000235:genetics; D014402:Tuberous Sclerosis / Q000235:genetics; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins
D012097:Repressor Proteins; C000624655:Tsc2 protein, rat; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins