Evaluation of oligonucleotide arrays for sequencing of the p53 gene in DNA from formalin-fixed, paraffin-embedded breast cancer specimens.
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BACKGROUND: Routine tissue processing has generated banks of paraffin-embedded tissue that could be used in retrospective cohort studies to study the molecular changes that occur during cancer development. The purpose of this study was to determine whether a p53 microarray could be used to sequence the p53 gene in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. METHODS: DNA was extracted from 70 FFPE breast cancer tissue specimens. p53 was sequenced with an oligonucleotide microarray (p53 GeneChip; Affymetrix), and the results were compared with the results obtained from direct sequencing. RESULTS: DNA was extracted from 62 of 70 cases. We identified 26 mutations in 24 of the 62 cases by the p53 GeneChip. No polymorphisms were detected, and exon 4 could not be evaluated in 20 cases. There were 43 genetic alterations detected by direct sequencing in 35 of the 62 cases. These consisted of 26 polymorphisms and 17 mutations in exons or splice sites. Fifteen mutations were identified by both methods. Direct sequencing detected significantly more gene alterations (43 of 54) in DNA extracted from FFPE tissue than the p53 GeneChip (26 of 54; P = 0.018). However, if the changes in exon 4 were eliminated from this comparison, the p53 GeneChip detected 26 of 27 mutations compared with direct sequencing, which identified 16 of 27 mutations. (P = 0.016). CONCLUSIONS: A combination of oligonucleotide microarray and direct sequencing may be necessary to accurately identify p53 gene alterations in FFPE breast cancer. The p53 GeneChip cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue. (+info)
Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.
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Little is known of molecular requirements for specification of human germ cells. However, it is likely that they are specified through the action of sequentially expressed genes just as in model organisms. We sought to determine whether human embryonic stem (ES) cell lines, like those of mice, might be capable of forming germ cells in vitro. We compared transcriptional profiles of three pluripotent human ES cells to those of isolated inner cell mass (ICM) cells. We found that ICM cells expressed NANOS1, STELLAR and OCT4, whereas undifferentiated human ES cells expressed these genes along with the germ cell-specific gene, DAZL. Upon ES cell differentiation into embryoid bodies (EBs), we observed a shift in expression from RNA and protein markers of immature germ cells to those indicative of mature germ cells, including expression of VASA, BOL, SCP1, SCP3, GDF9 and TEKT1. Although ability to test the function of these putative VASA positive germ cells is limited, these results demonstrate that differentiation of human ES cells into EBs in vitro results in formation of cells that express markers specific to gonocytes. (+info)
Anti-human Olig2 antibody as a useful immunohistochemical marker of normal oligodendrocytes and gliomas.
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Olig2 is a recently identified transcription factor involved in the phenotype definition of cells in the oligodendroglial lineage. The expression of Olig2 transcript has been demonstrated in human oligodendroglial tumors, although the protein expression has not been studied extensively. We developed a polyclonal antibody to human Olig2 and analyzed it immunohistochemically. The antibody depicted a single distinct band of predicted molecular weight by Western blotting, and did not cross-react with human Olig1. In normal human brain tissue, the nuclei of oligodendrocytes of interfascicular, perivascular, and perineuronal disposition were clearly labeled by the antibody. Similarly, the nuclei of oligodendroglial tumors were labeled. There was no apparent correlation between the staining intensity and histological grade. Astrocytic components within the tumors were generally less or not stained. Astrocytic tumors were also positive with the Olig2 antiserum to a lesser extent, and the difference between oligodendroglial and astrocytic tumors was demonstrated by a statistical analysis. Olig2 and glial fibrillary acidic protein were expressed in a mutually exclusive manner, and Olig2 expression was cell-cycle related. Neither central neurocytoma nor schwannoma cases were stained. Our antibody was demonstrated to be useful in recognizing normal oligodendrocytes on paraffin sections, and applicable in diagnosis of some brain tumors. (+info)
Rapid triple-labeling method combining in situ hybridization and double immunocytochemistry.
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A new, rapid method is described for combining in situ hybridization and immunocytochemistry to define cell populations and to map three-dimensional movements of groups of labeled cells within developing chick embryos. The method allows fluorescently labeled cells to be followed in living embryos and subsequently detected as a permanent reaction product for detailed three-dimensional analysis by immunocytochemistry in histological serial sections. Cell identity can be ascertained using a specific riboprobe and in situ hybridization. With this approach, the movements of two groups of cells can be mapped simultaneously (using two different fluorescent trackers and, subsequently, two different chromogens for immunocytochemistry) to analyze relative movements within an embryo, and when combined with in situ hybridization with a specific riboprobe for cell identity, allows fate mapping studies to be conducted using molecular criteria, rather than solely at morphological/positional criteria. The improved method enables the investigator to extract substantially more information from individual embryos, maximizing the results obtained from labor-intensive fate mapping studies. (+info)
Long-term preservation of antigenicity on tissue microarrays.
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Tissue microarrays have facilitated the evaluation of large cohort studies; however, there is little data on the best method for preserving sections once they are cut. We assessed three methods of storing precut breast cancer microarray slides: paraffin coating and storage in a nitrogen desiccator, either alone or in combination. We tested the durability of three antigens, cytokeratin, estrogen receptor, and Ki-67 on microarrays stored under these conditions for 3 months at room temperature. Staining was assessed with both manual scoring using traditional brown stain (0-3+) as well as automated scoring using fluorescently stained sections. Staining intensity was compared to that obtained from freshly cut slides. Slides stored under ambient conditions (room temperature and air) for 3 months exhibited marked degradation of all target antigens, in some cases resulting in slides that were virtually unreadable. We found that combined paraffin coating and nitrogen storage resulted in the best preservation of antigenicity, with retention of 72-99% of the antigenicity of a freshly cut slide, depending upon the marker and detection system used. The use of either paraffin coating or nitrogen storage alone protected slides to a lesser degree. (+info)
High-frequency oscillatory ventilation and an interventional lung assist device to treat hypoxaemia and hypercapnia.
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A male patient accidentally aspirated paraffin oil when performing as a fire-eater. Severe acute respiratory distress syndrome (Pa(o(2))/Fi(o(2)) ratio 10.7 kPa) developed within 24 h. Conventional pressure-controlled ventilation (PCV) with high airway pressures and low tidal volumes failed to improve oxygenation. Hypercapnia (Pa(co(2)) 12 kPa) with severe acidosis (pH<7.20) ensued. Treatment with high-frequency oscillatory ventilation (HFOV) and a higher adjusted airway pressure (35 cm H(2)O) improved the Pa(o(2))/Fi(o(2)) ratio within 1 h from 10.7 to 22.9 kPa, but the hypercapnia and acidosis continued. Stepwise reduction of the mean airway pressure (26 cm H(2)O), and oscillating frequencies (3.5 Hz), as well as increasing the oscillating amplitudes (95 cm H(2)O) resulted in an unchanged Pa(co(2)), but oxygenation worsened. The new pumpless extracorporeal interventional lung assist device (ILA, NovaLung, Hechingen, Germany) was therefore used for carbon dioxide elimination to enable a less aggressive ventilation strategy. Pa(co(2)) normalized after initiation of ILA. HFOV with a mean airway pressure of 32 cm H(2)O was maintained, but with a higher oscillatory frequency (9 Hz) and very low oscillatory amplitude (25 cm H(2)O). After 6 days, the patient was transferred to a conventional ventilator, and ILA was discontinued after 13 days without complications. (+info)
RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.
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In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results. (+info)
Comparison of snap freezing versus ethanol fixation for gene expression profiling of tissue specimens.
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Frozen tissue specimens are the gold standard for molecular analysis. However, snap freezing presents several challenges regarding collection and storage of tissue, and preservation of histological detail. We evaluate an alternative preservation method, ethanol fixation followed by paraffin embedding, by analyzing expression profiles of microdissected cells on Affymetrix oligonucleotide arrays of three matched benign prostatic hyperplasia (BPH) and tumor samples processed with each preservation method. Frozen samples generated an average present call of 26% of the probe sets, compared to 4.5% in ethanol-paraffin samples. Eighty-eight percent of the probe sets called present in the ethanol-paraffin samples were also present in the frozen specimens. Comparing ethanol-paraffin BPH to tumor, 52 probe sets showed a twofold differential expression or higher in at least two cases, 23 of which were also differentially expressed in at least one frozen case. Despite a significant drop in the number of transcripts detectable, the data suggests that the obtainable information in ethanol-fixed samples may be useful for molecular profiling where frozen tissue is not available. However, ethanol fixation and paraffin embedding of tissue specimens is not optimal for high-throughput mRNA expression analysis. Improved methods for transcript profiling of archival samples, and/or tissue processing are still required. (+info)