PCR artifacts in LOH and MSI analysis of microdissected tumor cells.
Polymerase chain reaction (PCR) analysis to study loss of heterozygosity (LOH) and microsatellite instability (MSI) in tumors is widely used. Microdissection techniques are applied to obtain tumor-specific tissue cells. By microdissection, however, the amount of template DNA extracted may vary considerably and interfere with optimal PCR amplification. To circumvent LOH and MSI misinterpretations due to low DNA input, we have assessed the critical level of DNA input for reliable PCR analysis. PCR analysis was performed by using 18 polymorphic markers (mono-, di-, tri-, and tetranucleotide) on DNA derived from both paraffin-embedded, formalin-fixed, and fresh frozen tumor specimens at template input levels ranging from 0.05 to 25.0 ng. We show a highly significant relation between DNA input and the occurrence of LOH and MSI artifacts. Furthermore, for DNA extracted from paraffin-embedded material, the percentage of LOH artifacts is significantly higher compared with DNA extracted from frozen tissue. For reliable PCR analyses using a mono-, di-, tri-, or tetranucleotide marker, a minimum of 10.0 ng DNA is required when DNA is isolated from formalin-fixed, paraffin-embedded tissue and 5.0 ng when isolated from fresh frozen tissue. HUM PATHOL 31:1414-1419.
Sieben
N L
NL
Department of Pathology, University Medical Center, Leiden, The Netherlands.
ter Haar
N T
NT
Cornelisse
C J
CJ
Fleuren
G J
GJ
Cleton-Jansen
A M
AM
D000483:Alleles; D016477:Artifacts; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D002277:Carcinoma / Q000235:genetics / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis*; D004210:Dissection; D005260:Female; D006801:Humans; D019656:Loss of Heterozygosity; D018442:Lymphoma, B-Cell, Marginal Zone / Q000235:genetics / Q000473:pathology; D008846:Micromanipulation; D018895:Microsatellite Repeats; D009369:Neoplasms / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology
D004273:DNA, Neoplasm