PCR artifacts in LOH and MSI analysis of microdissected tumor cells

Sieben, Nathalie L.G.; Haar, N. T. Ter; Cornelisse, Cees J.; Fleuren, Gebt Jan; Cleton-Jansensen, Anne-Marie

doi:10.1016/s0046-8177(00)80013-1

Cited by 83 publications

(51 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This minimized internal variability length among reference DNA and DNA from carcinomas. Samples were similar in magnitude and ranged from 500 to 1,000 cells in different cases, as indicated by Sieben et al [34].…”

Section: Oligonucleotide Cgh Microarrayssupporting

confidence: 63%

Genetic similarities and differences between lobular in situ neoplasia (LN) and invasive lobular carcinoma of the breast

et al. 2006

View full text Add to dashboard Cite

One of the most controversial issues in breast pathology is whether lobular neoplasia (LN) is a risk factor or a precursor lesion of invasive lobular carcinoma (ILC). This is consequent to the fact that no conclusive data on the biology of LN exist. Molecular studies of LN and ILC are scanty, variable, and not consistent. Clonality of 12 cases of LN and ILC present simultaneously in the same block has been studied. Cells from both lesions were obtained by microdissection and were studied for mitochondrial DNA (mtDNA), D-loop sequencing, and neighbor-joining trees. Eight of the same cases were studied with comparative genomic hybridization (CGH) array to have additional data consistent with mtDNA. In all cases, loss of heterozygosity was studied for D16S496,locus 16q22.1 related to e-cadherin. It appears that no fewer than eight cases were genetically very similar (clonal) with mtDNA. Seven of these cases appeared also clonal with CGH array. It is concluded that in the present series, LN and ILC are genetically related lesions in the majority of cases and that LN might be the precursor of ILC.

show abstract

Section: Oligonucleotide Cgh Microarrayssupporting

confidence: 63%

Genetic similarities and differences between lobular in situ neoplasia (LN) and invasive lobular carcinoma of the breast

et al. 2006

View full text Add to dashboard Cite

show abstract

“…First, because the DNA isolated by the microdissection technique from formalin-fixed tissues is considerably degenerated and exists at a low concentration, the PCR might be susceptible to frequent errors. In fact, Sieben et al 32 showed that MI artifacts were not encountered at a DNA concentration of 25ng per reaction volume, but that they were present in 16.5% of samples when 0.05ng of template DNA was used. Second, it is difficult to distinguish the "zebra-pattern" band of PCR artifacts from MI by conventional autoradiography.…”

Section: Discussionmentioning

confidence: 99%

Microsatellite instability and loss of heterozygosity in the nondysplastic colonic epithelium of ulcerative colitis

Takahashi

Kojima

Kinouchi

et al. 2003

Journal of Gastroenterology

View full text Add to dashboard Cite

Background. A major longterm risk for patients with ulcerative colitis (UC) is the development of colorectal dysplasia and cancer. Microsatellite instability (MI) has now been reported not only in colitic cancers but also in dysplasias, and even in nondysplastic inflamed mucosa. With the conventional microdissection technique, however, contamination by nonepithelial cells cannot be prevented and this could produce less reliable results than other methods. Therefore, we examined the condition of MI and loss of heterozygosity (LOH) of UC epithelium using a crypt isolation technique. Methods. One hundred and twenty-nine biopsy samples from 21 patients with UC were investigated for histology and microsatellite status, using nine microsatellite markers. A total of 1031 polymenase chain reaction (PCR) products were evaluated. Results. We found that no microsatellite markers displayed instability, but LOH at the 3p locus was detected in the nondysplastic epithelium of one patient with longstanding UC. Conclusions. Our study strongly suggests that MI does not contribute to the progression of the colitis-dysplasia sequence.

show abstract

“…To circumvent PCR artefacts [30], 10 ng/l purified template DNA was used in a 12-l reaction volume containing 6 pmol of each primer, 2 mM dNTPs, 0.1 mg/ml bovine serum albumin, Taq polymerase buffer (10 mM Tris-HCl, 1.5 mM MgCl 2 , 50 mM KCl, 0.01% [w/v] gelatin, 0.1% Triton), and 1.0 unit AmpliTaq Gold polymerase (Applied Biosystems Inc., Foster City, CA). The forward or reverse PCR primer was fluorescently labeled with FAM or TET, respectively.…”

Section: Loh Analysismentioning

confidence: 99%