Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique

Beers, Philip; Wittliff, James L.

doi:10.1016/0003-2697(75)90366-8

Cited by 19 publications

(4 citation statements)

References 18 publications

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“…The remaining cells were centrifuged at 300 ϫ g for 10 min at 4 Њ C, the supernatant was poured off, and 1 ml of Tri Reagent added for each 10 7 cells to lyse the cells, denature the proteins, and release nucleic acids. The solution then was either processed for RNA extraction or frozen at Ϫ 70 Њ C. RNA extraction and RT were carried out according to previously described methods (18,27) and methods described in the Tri Reagent manufacturer protocol (28). Frozen samples could be stored for as long as 72 h without loss of RNA.…”

Section: Sample Preparation and Rna Extractionmentioning

confidence: 99%

Quantitative RT-PCR Measurement of Cytochromes p450 1A1, 1B1, and 2B7, Microsomal Epoxide Hydrolase, and NADPH Oxidoreductase Expression in Lung Cells of Smokers and Nonsmokers

Willey

Coy²,

Frampton³

et al. 1997

Am J Respir Cell Mol Biol

125

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Bronchial epithelial cells (BEC) are the progenitors of bronchogenic carcinomas and are exposed to polycyclic aromatic hydrocarbon (PAH) procarcinogens through inhalation of combustion products. PAH are converted to carcinogenic molecules through a combination of monoxygenation by cytochrome p450 (CYP) enzymes in the presence of NADPH oxidoreductase (OR) and hydrolysis by microsomal epoxide hydrolase (mEH). In artificial systems, the relative expression of these genes determines whether carcinogenic or noncarcinogenic species are generated during metabolism. This relationship was explored in humans by using quantitative competitive reverse transcriptase polymerase chain reaction amplification to determine the range of expression of CYP1A1, CYP1B1, mEH, and NADPH OR in BEC recovered from 10 nonsmokers and 9 smokers. CYP2B7 expression was evaluated because, although little is known of its substrate specificity, it is expressed at high levels in human lung tissue. CYP1A1 and CYP1B1 were expressed in BEC at significantly different levels (P < 0.05) in the 9 smokers at 1.4 +/- 2.3 x 10(4) and 2.4 +/- 3.2 x 10(3) molecules/10(6) beta-actin molecules (mean +/- STD), respectively, but each was measurable in only one of the 10 nonsmokers. There was significant inter-individual variation (P < 0.05) in both CYP1A1 and CYP1B1 expression among the subjects for whom sufficient data were obtained. The inducibility of human BEC CYP1A1 gene by PAH exposure was confirmed in vitro by incubating cultured immortalized human BEC with beta-naphthoflavone and observing a > 6-fold induction of CYP1A1 after 24 h. In contrast to BEC, alveolar macrophages expressed CYP1A1 at low (30-70 molecules/10(6) beta-actin molecules) to unmeasurable levels in both smokers and nonsmokers. There was no significant difference in expression of mEH, CYP2B7, or NADPH OR in smokers compared with nonsmokers. The inter-individual variation in absolute and relative expression of PAH metabolism enzymes in BEC reported here supports the hypothesis that inter-individual variation in ability to activate/inactivate inhaled PAH carcinogens accounts for at least some of the inter-individual variation in risk for bronchogenic carcinoma.

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Section: Sample Preparation and Rna Extractionmentioning

confidence: 99%

Quantitative RT-PCR Measurement of Cytochromes p450 1A1, 1B1, and 2B7, Microsomal Epoxide Hydrolase, and NADPH Oxidoreductase Expression in Lung Cells of Smokers and Nonsmokers

Willey

Coy²,

Frampton³

et al. 1997

Am J Respir Cell Mol Biol

125

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“…Firstly, a cell-lysing solution at a high salt concentration was used to dissociate and denature proteins (apart from DNase and RNase) bound to DNA and RNA (LePecq and Paoletti, 1966), and to obtain optimal binding of ethidium bromide to the intercalated site that is specific for double-stranded polynucleotides Paoletti, 1966, 1967). EDTA was added to the lysing solution to prevent DNase action (Beers and Wittliff, 1975). After cell lysis, heparin was added in order to displace DNA from nuclear proteins and to form complexes with these nuclear proteins (Karsten and Wollenberger, 1977).…”

Section: Discussionmentioning

confidence: 99%

“…They also observed that EB binds quantitatively to DNA and RNA (LePecq and Paoletti, 1967) at PH 8.0. Thus, DNA and RNA contents of cells or tissues may be determined (Beers and Wittliff, 1975). This is a sensitive method, since 0.01 pg/ml of DNA can still be detected (LePecq and Paoletti, 1966).…”

mentioning

confidence: 99%

Detection of DNA cross‐links in tumor cells with the ethidium bromide fluorescence assay

Jong¹,

Zijlstra²,

Timmer‐Bosscha³

et al. 1986

Intl Journal of Cancer

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Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single- and double-stranded DNA (dsDNA). Cross-links in DNA are measured by the return of fluorescence of dsDNA after heat denaturation at pH 12. Under these conditions denatured DNA gives very low fluorescence. In the present study this assay was applied to tumor cells. The mouse Ehrlich ascites tumor cell line (EAT) and a human small-cell carcinoma line (GLC4) were incubated for 4 hr at 37 degrees C, with the cross-linking agent cis-diamino-dichloro platinum (cDDP). The samples of whole cells were thereafter resuspended in potassium phosphate buffer with 10 mM EDTA, 4M NaCl, 0.1% Sarkosyl pH 7.2, for 16 hr at 37 degrees C. Measurements were performed with a spectrofluorometer with excitation wavelength 525 nm, emission wavelength 580 nm. There was a linear relationship for cDDP concentrations of 0-150 microM and the extent of DNA cross-links in EAT (r = 0.958). In GLC4 there was a linear relationship at low cDDP concentrations of 0-50 microM (r = 0.968) while between 50 and 150 microM a plateau was reached. RNase added to the lysate of whole cells had no influence on the extent of cross-links. This assay was compared with the alkaline elution assay, and results were identical.

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“…They suggested that this binding could be used as the basis of an assay for RNA, and Beers & Wittliff (1975) have shown that using ethidium bromide it is possible to assay RNA (mainly ribosomal) along with DNA, although their extraction of RNA is inefficient and the method is as slow as and less sensitive than current methods for measuring RNA. The fluorescent enhancement of ethidium bromide on binding has also been used to investigate ribosomal structure (Bollen et al, 1970;Gatti et al, 1977).…”

mentioning

confidence: 99%

A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations

Mackie¹,

Bryant²,

Mowbray³

1979

Biochemical Journal

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Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique

Cited by 19 publications

References 18 publications

Quantitative RT-PCR Measurement of Cytochromes p450 1A1, 1B1, and 2B7, Microsomal Epoxide Hydrolase, and NADPH Oxidoreductase Expression in Lung Cells of Smokers and Nonsmokers

Quantitative RT-PCR Measurement of Cytochromes p450 1A1, 1B1, and 2B7, Microsomal Epoxide Hydrolase, and NADPH Oxidoreductase Expression in Lung Cells of Smokers and Nonsmokers

Detection of DNA cross‐links in tumor cells with the ethidium bromide fluorescence assay

A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations

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