High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

Lin, Jianghai; Kennedy, Stephen; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W.; Xu, Anlong; Zondervan, Krina T.

doi:10.1016/j.ab.2009.08.016

Cited by 99 publications

(70 citation statements)

References 10 publications

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“…Of these, 38 cases had characteristic histologic lesions but only 22 samples (individual tissue sections) that were either PCR positive and/or had nematodes present in histologic section were included in further study (Table 1). In 9 elk, 2 samples were from spinal cord and the other 8 were from brain (10). In alpacas, 3 samples were from spinal cord and 3 were from the brain (6).…”

Section: Resultsmentioning

confidence: 99%

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Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates

et al. 2014

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Abstract. Formalin-fixed, paraffin-embedded tissues from elk (Cervus elaphus), goats, and camelids with case histories and lesions suggestive of Parelaphostrongylus tenuis were examined by histology to characterize lesions that could aid in definitively diagnosing P. tenuis infection. Additionally, sections of paraffin-embedded tissue were used in a nested polymerase chain reaction (nPCR) using Parelaphostrongylus-specific primers to determine how PCR results corresponded with histological findings. Histological changes in brain and spinal cord consisted of linear tracks of hemorrhage; tracks or perivascular accumulations of hemosiderin-laden macrophages; acute foci of axonal degeneration and/or linear glial scars; and perivascular, parenchymal, or meningeal accumulations of eosinophils and/or lymphocytes and plasma cells. Of the 43 samples with histologic lesions consistent with neural larval migrans, 19 were PCR positive; however, only 8 were confirmed Parelaphostrongylus by DNA sequencing. Additionally, 1 goat was identified with a protostrongylid that had a 97% identity to both Parelaphostrongylus odocoilei and a protostrongylid nematode from pampas deer (Ozotoceros bezoarticus celer) from Argentina. None of the histologic lesions individually or in combination correlated statistically to positive molecular tests for the nematode. The results indicate that it is possible to extract Parelaphostrongylus DNA from formalin-fixed, paraffinembedded tissue, but extended fixation presumably can cause DNA crosslinking. Nested PCR provides another diagnostic tool to identify the cause of neurologic disease in camelids and elk with histologic lesions consistent with neural larval migrans. Furthermore, potential novel protostrongylid DNA was detected from a goat with lesions consistent with P. tenuis infection, suggesting that other neurotropic Parelaphostrongylus species may occur locally.

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Section: Resultsmentioning

confidence: 99%

“…[3][4][5]10,13,18 Using nPCR allows for increased sensitivity to detect Parelaphostrongylus spp. DNA at quantities that conventional PCR would not detect.…”

Section: Research-article2014mentioning

confidence: 99%

Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates

et al. 2014

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“…By comparing our DNA isolation strategy with those described in previous studies (Lin et al, 2009;Santos et al, 2009;Farrugia et al, 2010;Okello et al, 2010), our results indicate that the method of stocking frozen formalin fixation tissues for long periods of time is the major factor affecting the quality of genomic DNA. The pretreatment of the formalin fixation tissues by gradual dehydration can effectively reduce the crosslinking between histone proteins and DNA, even though formaldehyde cannot be completely removed (Fang et al, 2002).…”

Section: Discussionmentioning

confidence: 97%

Improved protocol for extracting genomic DNA from frozen formalin-fixed tissue resulting in high-quality whole mtDNA

Wang

Zhang

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Formalin fixation and paraffin embedding is widely used for convenient and long-term storage of tumor tissue and precious sources to perform genetic studies. However, DNA fragmentation is one of the major flaws of genomic DNA isolation from formalin fixation tissues, which limits its further usage. Here, we present an improved method for isolating high-quality genomic DNA from formalin fixation tissue. We obtained high-quality genomic DNA of more than 20 kb from samples frozen for more than 2 years. Furthermore, to verify DNA quality, the whole mitochondrial DNA (mtDNA) genomes from the normal and tumor tissue of the same patient were successfully amplified with two overlapping PCR fragments comprising more than 8379 bp in length for each fragment. In addition, the whole genomes were sequenced with a 48-well based primer panel in order to avoid potential sequencing errors from artificial recombination, which was further confirmed with an mtDNA phylogenetic strategy. Our improved DNA extraction method from formalin fixation tissue and sequencing strategy for entire mtDNA genomes will generate unambiguous sequence analysis results for clinical samples.

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“…2 In particular, the presence of formaldehyde induces crosslinking between nucleic acids and proteins, and the low pH (<1) induces fragmentation. 3,4 Nevertheless, FFPE-archived tissues are more easily accessible than fresh or frozen tissues, thus much effort has been made to use FFPE tissues for different genetic analysis.…”

Section: Introductionmentioning

confidence: 99%

Analysis of mRNA Expression Levels in FFPE Testicular Biopsies Using Real-time PCR Arrays

2016

ERHM

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Background and objective: Tissues archived for long-term storage as formalin-fixed, paraffin-embedded (FFPE) samples represent rich source of material for genomic studies, but the nucleic acid isolation and downstream analysis is technically difficult. This is especially true when conducting mRNA expression studies, which strongly depend on the quality and quantity of the starting material for successful data normalization. Our objective was to investigate the mRNA expression levels in testicular FFPE samples using realtime PCR arrays and to present our experience with some technical challenges. Methods: Total RNA was extracted from FFPE samples from six patients with hypospermatogenesis and three controls using the Qiagen AllPrep DNA/RNA FFPE Kit. The integrity of the isolated RNA was assessed by an Agilent Bioanalyzer 2100. Qiagen Human Male Infertility RT 2 Profiler PCR Arrays were used to study the expression of 84 genes. Results: Our experience with the analysis of the FFPE testicular samples using the RT 2 Profiler PCR Array showed difficulties with the data normalization, despite using the same amount of starting material and similar values for the RNA integrity numbers (RINs) across all the samples, as recommended by the manufacturer. By using the percentage of the relatively intact RNA (area between 150 bp and 4,000 bp on the electropherograms of the Bioanalyzer 2100), we observed a negative correlation between amount of the intact RNA and average Ct values of the analyzed genes. Our initial results using PCR Array analysis on FFPE testicular tissues revealed 38 differentially expressed genes that

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High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

Cited by 99 publications

References 10 publications

Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates

Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates

Improved protocol for extracting genomic DNA from frozen formalin-fixed tissue resulting in high-quality whole mtDNA

Analysis of mRNA Expression Levels in FFPE Testicular Biopsies Using Real-time PCR Arrays

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