Loss of Heterozygosity Studies Revisited

Abstract

Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) studies of archival formalin-fixed, paraffin-embedded (FFPE) tumor tissues have become an important tool in the search for tumor suppressor genes and oncogenes and are also used increasingly in clinical practice. However, FFPE tissue samples may contain little amplifiable DNA, resulting in frequent reaction failures and unreliable LOH data. Using pairs of serial dilutions of reference DNA, we determined the minimum amplifiable DNA concentration necessary for reliable microsatellite-PCR LOH analysis. We then measured the amplifiable DNA content of a selection of frozen and FFPE-derived tumor specimens by real-time quantitative PCR. A minimum input of 600 pg of 100% amplifiable DNA per PCR was required for reliable LOH analysis. While the total DNA concentrations of all samples exceeded this figure, most FFPE-sample-derived DNA was non-amplifiable, with ratios of actually amplifiable DNA to total DNA as low as 1 to 3625. Many FFPE samples therefore contained substantially less than 600 pg/microl of actually amplifiable DNA, making them potentially unsuitable for LOH studies. Real-time quantitative PCR before LOH studies of FFPE tissues allows: identification of samples, which will fail microsatellite-PCR; exclusion of samples, which will yield unreliable results; and optimal adjustment of template input for the remainder. Amplification reactions undertaken without this precaution can result in unreliable LOH data.

Molecular Heterogeneity in Primary Breast Carcinomas and Axillary Lymph Node Metastases Assessed by Genomic Fingerprinting Analysis

“…Limitations to our study included the small number of patients with sufficient tissue from primary tumors and LN metastases available for analysis, which limited the conclusions that could be drawn from the data. Although a number of steps were taken to ensure the accuracy of AI detection, 32 DNA from FFPE samples is prone to technical artifact, 33 , 34 and thus may not accurately estimate the number of genetic alterations. Given the small areas of tumor sampled, we examined hypervariable STR regions representing a small portion of the genome to derive molecular fingerprints of these areas and did not use global technologies such as comparative genomic hybridization or next-generation sequencing.…”

Manipulating the Salt and Thermal Stability of DNA Multilayer Films via Oligonucleotide Length

“…A fixed number of DNA-coated particles (2 × 10 6 particles per mL) suspended in 3 mL of SSC buffer was incubated with 3 μL of a 100-fold diluted picogreen solution for 3 min prior to the measurements. The DNA melt curves were obtained by monitoring the fluorescence emission of picogreen at 520 nm over a range of temperatures . When a Peltier heating device was used, the temperature of the suspension was ramped up in 1 °C increments with a 0.5 min equilibrium time over the temperature range of 20 to 80 °C to obtain the first melt curve, T m 1 .…”

“…The DNA melt curves were obtained by monitoring the fluorescence emission of picogreen at 520 nm over a range of temperatures. 39 When a Peltier heating device was used, the temperature of the suspension was ramped up in 1 °C increments with a 0.5 min equilibrium time over the temperature range of 20 to 80 °C to obtain the first melt curve, T m1 . The suspension was cooled down to room temperature to allow rehybridization of the denatured strands in the solution.…”

Microarray-based DNA methylation study of Ewing’s sarcoma of the bone

“…All DNA samples were stored at −70°C prior to use. The DNA concentrations derived from the FFPE samples were determined on a fluorophotometer (Victor3; Perkin-Elmer, Waltham, MA, USA), using the PicoGreen nucleic acid quantification kit (PicoGreen; Molecular Probes, Eugene, OR, USA), which allows accurate and reproducible DNA quantification at low concentrations, including DNA extracted from archival FFPE samples ( 14 ).…”