Bisulfite Conversion of DNA from Tissues, Cell Lines, Buffy Coat, FFPE Tissues, Microdissected Cells, Swabs, Sputum, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

2015 ◽  
pp. 139-159 ◽  
Author(s):  
Maria Jung ◽  
Barbara Uhl ◽  
Glen Kristiansen ◽  
Dimo Dietrich
Keyword(s):  
Cell Lines ◽  
Buffy Coat ◽  
Bisulfite Conversion ◽  
Ffpe Tissues ◽  
Serum And Urine

scholarly journals Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

PLoS ONE ◽  
2014 ◽  
Vol 9 (4) ◽  
pp. e93933 ◽  
Author(s):  
Emily Eva Holmes ◽  
Maria Jung ◽  
Sebastian Meller ◽  
Annette Leisse ◽  
Verena Sailer ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Cell Lines ◽  
Bisulfite Conversion ◽  
Ffpe Tissues ◽  
Serum And Urine

Quantification of HER2 from gastroesophageal cancer (GEC) FFPE tissue by mass spectrometry (MS).

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 17-17 ◽  
Author(s):  
Todd A. Hembrough ◽  
Les Henderson ◽  
Brittany Rambo ◽  
Wei-Li Liao ◽  
Sheeno Thyparambil ◽  
...  
Keyword(s):  
Cell Lines ◽  
Her2 Status ◽  
Molecular Heterogeneity ◽  
Her2 Expression ◽  
Her2 Positive ◽  
Ffpe Samples ◽  
Ffpe Tissues ◽  
Pc3 Cells ◽  
One Year ◽  
Sensitivity Specificity

17 Background: Trastuzumab had a survival benefit in ‘HER2 positive’ GEC, determined by IHC/FISH. These companion diagnostics have limitations. IHC is semi-quantitative, subjective, and sensitive to antigen instability in FFPE; FISH is laborious, expensive, and subjective. Gene amplification may not correlate to protein expression. Moreover these are low throughput assays. There is increased recognition of profound interpatient molecular heterogeneity with several putative biomarkers, and only scarce tissue to assess for each one. We sought to evaluate our MS platform on GEC FFPE tissues for HER2 status compared to IHC/FISH. We also applied the “GEC-plex” of 11 other potentially predictive/prognostic markers for GEC. Methods: We utilized trypsin digestion mapping of rHER2 to identify unique peptides for SRM development. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in a complex eukaryotic matrix (PC3 cells) to determine LOD, LLOQ, accuracy, precision and linearity of the assays. The assay was run on 17 GEC cell lines, in parallel with FISH/IHC, and expression thresholds were established for HER2+/HER2-; the sensitivity/specificity of the established cutoffs were then tested prospectively in FFPE GEC tissues on 10uM FFPE LCM slides (n=121). HER2 stability from FFPE sections was assessed by assaying 33 freshly cut FFPE samples; the adjacent sections were processed one year later. Results: The HER2 peptide chosen (ELVSEFSR) had a LOD of 100 amol and CV<20%. HER2 MS on GEC cell lines revealed concordance with FISH (HER2:CEP17) ratio (R2=0.96). The analysis suggested HER2 expression > 750 amol/ug was indicative of HER2 amplification. The assay was stable in archival FFPE sections (R2=0.76). For GEC FFPE cases, ‘any’ HER2 expression was seen in 69.4% of cases; 8.2% showed HER2 > 750 amol (10/121) - all were HER2 amplified. No cases <550 amol/ug were HER2 amplified. IHC/FISH results for cases with 550-750 amol/ug demonstrated a heterogeneous ‘equivocal’ zone, not unlike ‘IHC 2+’, which may require FISH confirmatory testing. Conclusions: ‘GEC-plex’ has a quantitative, sensitive, and specific HER2 assay that can be multiplexed along with other GEC biomarkers.

Quantification of MET expression using mass spectrometry (MS): Assay precision and stability in FFPE tumor tissue.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 16-16 ◽  
Author(s):  
Todd A. Hembrough ◽  
Wei-Li Liao ◽  
Sheeno Thyparambil ◽  
Les Henderson ◽  
Brittany Rambo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Temporal Stability ◽  
Therapeutic Benefit ◽  
Test Methods ◽  
Cross Reactivity ◽  
Selected Reaction Monitoring ◽  
Ffpe Tumor ◽  
Ffpe Samples ◽  
Ffpe Tissues ◽  
One Year

16 Background: Overexpression of MET in gastroesophageal cancer (GEC) is associated with poor prognosis and potentially predictive of anti-MET therapeutic benefit. IHC has been the method chosen to quantify MET expression to date. However, IHC is semi-quantitative, suffers from cross-reactivity, and is low throughput. Moreover, MET IHC is hampered by antigenic instability in FFPE sections, limiting its utility to recently cut FFPE sections. Increasing recognition of the importance of other biomarkers in GEC suggests that ‘economic’ testing of scarce samples will be required. We sought to develop a MET quantitative assay within our ‘GEC-plex’ Liquid-Tissue-selected reaction monitoring (LT-SRM) MS test. Methods: We used trypsin digestion mapping of rMET to identify unique peptides for MS assay development. The assay was pre-clinically validated in 5 cell lines, where electrochemiluminescence (ECL) immunoassay measurement of MET was also performed. To assess the MET MS assay stability from archival FFPE sections, freshly cut FFPE tissue sections were immediately microdissected, processed and analyzed, while adjacent sections were processed and analyzed one year after cutting, to compare temporal quantification from the same FFPE samples (n=33). MET expression was assessed in GEC cases (n=121), and compared to IHC and FISH in select cases. Results: Tryptic digestion mapping of rMET showed that peptide TEFTTALQR was optimal for MET quantification. The LLOD for this peptide was 150 amol with CV<20%. Validation of the MET MS assay on 5 cell lines revealed concordance when compared to ECL (R2=0.99). The MET MS assay demonstrated temporal stability of serial sections cut from 33 samples analyzed one year apart: CVs<20%, R2=0.75. Analysis of 121 GEC FFPE tissues showed a broad range of MET expression levels (<150-4600 amol/ug), with 36/121 (29.7%) having detectable levels, similar to that observed using IHC. MS expression thresholds were determined that reliably identified MET gene amplification; sensitivity and specificity of these thresholds will be presented. Conclusions: ‘GEC-plex’ has a quantitative, sensitive, and specific MET MS assay that can be multiplexed along with other GEC biomarkers.

scholarly journals Increased MiR-221 expression in hepatocellular carcinoma tissues and its role in enhancing cell growth and inhibiting apoptosis in vitro

BMC Cancer ◽  
2013 ◽  
Vol 13 (1) ◽  
Author(s):  
Minhua Rong ◽  
Gang Chen ◽  
Yiwu Dang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
Cell Growth ◽  
Cell Lines ◽  
Clinicopathological Parameters ◽  
Clinical Samples ◽  
Therapy Strategy ◽  
Ffpe Tissues ◽  
The Relationship

Abstract Background MiR-221 is over-expressed in human hepatocellular carcinoma (HCC), but its clinical significance and function in HCC remains uncertain. The aim of the study was to investigate the relationship between miR-221 overexpression and clinicopathological parameters in HCC formalin-fixed paraffin-embedded (FFPE) tissues, and the effect of miR-221 inhibitor and mimic on different HCC cell lines in vitro. Methods MiR-221 expression was detected using real time RT-qPCR in FFPE HCC and the adjacent noncancerous liver tissues. The relationship between miR-221 level and clinicopathological features was also analyzed. Furthermore, miR-221 inhibitor and mimic were transfected into HCC cell lines HepB3, HepG2 and SNU449. The effects of miR-221 on cell growth, cell cycle, caspase activity and apoptosis were also investigated by spectrophotometry, fluorimetry, fluorescence microscopy and flow cytometry, respectively. Results The relative expression of miR-221 in clinical TNM stages III and IV was significantly higher than that in the stages I and II. The miR-221 level was also upregulated in the metastatic group compared to the nonmetastatic group. Furthermore, miR-221 over-expression was related to the status of tumor capsular infiltration in HCC clinical samples. Functionally, cell growth was inhibited, cell cycle was arrested in G1/S-phase and apoptosis was increased by miR-221 inhibitor in vitro. Likewise, miR-221 mimic accelerated the cell growth. Conclusions Expression of miR-221 in FFPE tissues could provide predictive significance for prognosis of HCC patients. Moreover, miR-221 inhibitor could be useful to suppress proliferation and induce apoptosis in HCC cells. Thus miR-221 might be a critical targeted therapy strategy for HCC.

Quantitative measurement of HER2 expression and HER2 homo-dimerization in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues using a novel proximity-based assay

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21023-21023
Author(s):  
W. Huang ◽  
Y. Tan ◽  
X. Jin ◽  
A. Mukherjee ◽  
J. Sperinde ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Gene Amplification ◽  
Dynamic Range ◽  
Predictive Test ◽  
Breast Cancers ◽  
Her2 Gene ◽  
Wide Dynamic Range ◽  
Her2 Gene Amplification ◽  
Ffpe Tissues

21023 Background: The best method to assess HER2 status remains controversial. Here we describe a novel assay that provides reliable quantitation of total HER2 (H2T) and HER2 homodimer (H22D) in FFPE tissues. The assay was cross-validated with conventional HER2 tests - immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Characteristics of the new assay and its potential application as predictive test were explored. Methods: Electrophoretic Tag (eTag) is a proprietary proximity-based technology that measures gene and protein expression. We have developed a novel application of the technology to quantify H2T and H22D in FFPE tissues. The validity of the assay was evaluated by analyzing its performance in cell lines and FFPE breast cancers. IHC (clone CB-11, Ventana) was performed in 174 breast cancers, scored according to standard IHC criteria and Histoscore (H-score), and HER2 gene amplification was evaluated by FISH (PathVysion, Vysis) in 19 breast cancers. eTag assays were performed on the same specimens and the results were compared with those of IHC and FISH. Results: H2T and H22D were proportional to the known expression levels in cell lines. H2T demonstrated a continuous measurement over a wide dynamic range (>2 logs), in contrast with conventional IHC (0, 1+, 2+, 3+). The correlation between H2T and IHC categories was significant (correlation=0.84, p<0.0001 by the Jonckheere-Terpstra test for trend), but overlap of H2T values in adjacent IHC categories was observed. H-score showed good correlation within the lower range of H2T, but plateaued at higher range of H2T. HER2 gene amplification (HER2/CEP17 ratio and gene copy #) by FISH was generally correlated with IHC categories and H2T values. There was a good correlation between H22D and H2T (r2= 0.7, P < 0.001). Conclusions: eTag reliably measures H2T and H22D in FFPE tissues. The continuous measure of H2T over a wide dynamic range and the novel H22D measure provide data superior to conventional HER2 tests by IHC and FISH, but clinical utility of the eTag assay remains to be investigated. eTag has potential as a predictive test for targeted HER2 therapy in breast cancer. No significant financial relationships to disclose.

(EB) Virus: Latency and Expression in Transplanted and Cultured Human Cells

1971 ◽  
Vol 29 ◽  
pp. 368-369
Author(s):  
B. G. Uzman ◽  
M. M. Kasac ◽  
H. Saito ◽  
A. Krishan
Keyword(s):  
Cell Lines ◽  
Primary Cultures ◽  
Virus Particles ◽  
Barr Virus ◽  
Virus Latency ◽  
Virus Surveillance ◽  
Cultured Human Cells ◽  
Epstein Barr ◽  
Serial Transplantation ◽  
Tubular Arrays

In conjunction with the cultivation and transplantation of cells from human tumors by the Programs of Microbiology and Immunogenetics, virus surveillance by electron microscopy has been routinely employed. Of particular interest in this regard have been 3 cell lines cultured from lymph nodes or spleen of 2 patients with Hodgkin's disease and 1 patient with Letterer-Siwe's disease. Each of these cell lines when transplanted in Syrian hamster neonates conditioned with anti-lymphocyte serum grew as serially transplantable tumors; from such transplants of the 3 cell lines cell cultures were retrieved.Herpes type virus particles (Figs. 1, 2, 3) were found in the primary cultures of all three lines, in frozen thawed aliquots of same, and in cultures retrieved from their tumors growing by serial transplantation in hamsters. No virus was detected in sections of 25 of the serially transplanted tumors. However, in 10 such tumors there were repeated instances of tubular arrays in the cisternae of the endoplasmic reticulum (Fig. 4). On serologic examination the herpes virus was shown to be the Epstein-Barr virus.

Comparison of Choriocarcinoma and Normal Placenta

1971 ◽  
Vol 29 ◽  
pp. 324-325
Author(s):  
John C. Garancis ◽  
Roland A. Pattillo ◽  
Robert O. Hussa ◽  
Jon V. Straumfjord
Keyword(s):  
Cell Lines ◽  
Small Cells ◽  
Tissue Cultures ◽  
Glycogen Depletion ◽  
Cell Surfaces ◽  
Intercellular Spaces ◽  
Concomitant Increase ◽  
Gestational Choriocarcinoma ◽  
Cytoplasmic Glycogen ◽  
Normal Placenta

Two different cell lines (Be-Wo and Jar) of human gestational choriocarcinoma have been maintained in continuous tissue culture for a period of four and two years respectively without losing the ability to elaborate human chorionic gonadotropin (HCG). Tissue cultures, as revealed by electron microscopy, consisted of small cells with single nuclei. In some instances cell surfaces were provided with microvilli but more often the intercellular spaces were narrow and bridged by desmosomes. However, syncytium was not formed. Endoplasmic reticulum (ER) was poorly developed in both cell lines, except in some Be-Wo cells it was prominent. Golgi complex, lysosomes and numerous free ribosomes, as well as excessive cytoplasmic glycogen, were present in all cells (Fig. 1). Glycogen depletion and concomitant increase of ER were observed in many cells following a single dose of 10 ugm/ml of adrenalin added to medium (Fig. 2).

Ultrastructure of rat alveolar macrophages activated in situ by poly:IC

1987 ◽  
Vol 45 ◽  
pp. 768-769
Author(s):  
A. M. Klinkner ◽  
R. A. Weiss ◽  
A. Kelley ◽  
P. J. Bugelski
Keyword(s):  
Venous Blood ◽  
Intratracheal Instillation ◽  
Buffy Coat ◽  
Fischer 344 Rats ◽  
Blood Monocytes ◽  
Fischer 344 ◽  
Polyinosinic:Polycytidylic Acid ◽  
Macrophage Activator ◽  
Endogenous Peroxidase

Polyinosinic:polycytidylic acid is an inducer of interferon and a macrophage activator. We have found that intratracheal instillation of polyI:C (IT-pI:C) activates rat bronchoalveolar lavage cells (BAL) for a variety of functions. Examination of Giemsa stained, cytocentrifuge preparations showed that IT-pI:C induced a population of BAL not seen in resident BAL. The morphology of these cells suggested that they might be derived from blood monocytes. To test this hypothesis we have examined several populations of macrophages that had been stained for endogenous peroxidase activity as a marker of cells derived from the monocyte-macrophage lineage.Macrophages were obtained from Fischer 344 rats. Peritoneal exudate cells (PEC) were collected by lavage 4 days after i.p. injection of 20 ml 3% thioglycolate. Buffy coat monocytes were separated from venous blood from naive rats.

Immunocytochemical localization of β1-4 galactosyltransferase in endometrial carcinoma cells

1990 ◽  
Vol 48 (3) ◽  
pp. 944-945
Author(s):  
Ichiro Yamamoto ◽  
Toshiaki Tachibana ◽  
Hiroko Maruyama ◽  
Noriyuki Komatsu ◽  
Hiroyuki Kuramoto ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Cell Lines ◽  
Endometrial Adenocarcinoma ◽  
Protein A ◽  
Rabbit Antiserum ◽  
Carcinoma Cells ◽  
Affinity Column ◽  
Antibody Technique ◽  
Galactosyl Transferase ◽  
C Terminus

We have paid attention to the alteration of glycosyltransferase in carcinoma cells, because it might be related to the malignancy of the cells. In this connection, localization of β1-4 galactosyl transferase (β1-4 Gal T) in human endometrial carcinoma cells was examined immunocytochemically using two kinds of cell lines, each of which showed different degree of differentiation.An antibody was purified from the rabbit antiserum against the synthetic peptide, IFNRLVFRGMSC (W89) of human β1-4 Gal T coupled with KLH (keyhole limpet hemocyanine) by protein A column and peptide-affinity column chromatography. The anti-W89 serum reacts to the C-terminus of human β 1-4 Gal T and to both membrane-bound and soluble forms of the enzyme. Cell line of well differentiated endometrial adenocarcinoma (I) and that of poorly differentiated endometrial adenocarcinoma (50B) were cultivated respectively in MEM medium containing 15% FCS and 2 mM glutamine for 4 d at 37°C under 5% CO2. The cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer (pH 7.4) at 4°C for 30 min, washed with PBS, then freezed and thawed. The indirect method of the peroxidase- labeled antibody technique was used for immunocytochemistry of both LM and TEM on the cell lines. The cells were dehydrated in ethanol and embedded in TAAB 812. Ultrathin sections were observed under a TEM, JEM-100S.

Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

1996 ◽  
Vol 54 ◽  
pp. 758-759
Author(s):  
D. W. Fairbain ◽  
M.D. Standing ◽  
K.L. O'Neill
Keyword(s):  
Heat Shock ◽  
Cell Lines ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Resistant Cell ◽  
Membrane Blebbing ◽  
Late Loss ◽  
Induced Apoptosis ◽  
Dying Cells ◽  
In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

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