A Branched DNA Signal Amplification Assay to Quantitate Messenger RNA of Human Uncoupling Proteins 1, 2, and 3

Zhou, Lubing; Cryan, Ellen V.; Minor, Lisa; Gunnet, Joseph W.; Demarest, Keith T.

doi:10.1006/abio.2000.4587

Cited by 15 publications

(6 citation statements)

References 22 publications

(27 reference statements)

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“…Another established high-throughput method for RNA quantification is the branched-chain DNA (bDNA) technology. 16 The b-DNA method is based on signal amplification in contrast to the target amplification employed in RT-PCR. The analysis can be performed directly on the cells, without RNA purification.…”

Section: Resultsmentioning

confidence: 99%

Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists

Rosengren

Simko

Aryan³

et al. 2005

SLAS Discovery

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The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor-1 (IGF-1). The liver is the main source of circulating IGF-I. The authors have used rodent primary hepatocytes for studies on pharmacological intervention of IGF-I mRNA expression. A 96-well nonradioactive IGF-1 mRNA quantification assay was developed, based on the hybridization of sense and antisense RNA probes, to replicate membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GH-receptor binding compounds were evaluated. Two compounds were found to down-regulate IGF-I mRNA. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate potential unspecific transcriptional effects, the mRNA levels of the housekeeping genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochrome P450 2C12 (CYP2C12) and a rat homologue to the human α1B-glycoprotein (A1BG), were quantified by RNase protection assays and found to be down-regulated, confirming the antagonistic property of 1 compound. In conclusion, a direct filter hybridization assay of hepatocyte lysates using nonradioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds. (Journal of Biomolecular Screening 2005:260-269)

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Section: Resultsmentioning

confidence: 99%

Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists

Rosengren

Simko

Aryan³

et al. 2005

SLAS Discovery

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“…We compared mRNA levels of treated cells to levels in vehicle control cells using a novel method of analysing mRNA levels in primary human hepatocytes based on the QuantiGene kit from Bayer, which may be as sensitive as RT-PCR for mRNA quantification (Hartley & Klaassen 2000;Warrior et al 2000;Zhou et al 2000;Sen et al 2001). The kit has an ELISA-like format and provides a solid support phase which binds RNA via probes specific for the gene of interest (capture extenders).…”

Section: Discussionmentioning

confidence: 99%

“…Cell lysates (90 ·L) were transferred to capture wells containing 100 ·L capture buffer provided with the kit, and wells were incubated overnight at 53¯C. For the remainder of the experiment, the instructions provided with the kit were closely followed, as described in Zhou et al (2000). Luminescence from the capture wells was measured on a luminometer (Wallac VICTOR 2 ; Perkin Elmer Life Sciences).…”

Section: Mrna Quantification (Branched Dna Assay)mentioning

confidence: 99%

Effect of bupropion on CYP2B6 and CYP3A4 catalytic activity, immunoreactive protein and mRNA levels in primary human hepatocytes: comparison with rifampicin

Hesse

Sakai

Vishnuvardhan

et al. 2003

Journal of Pharmacy and Pharmacology

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Animals treated with multiple doses of bupropion have had increased bupropion clearance or increased liver weight, suggesting induction of drug-metabolizing activity. The possibility of cytochrome p450 (CYP) induction by bupropion (10 microM) was evaluated in-vitro by comparing catalytic activity, immunoreactive protein and CYP mRNA levels from human hepatocytes in primary culture versus cells treated with vehicle (0.5% methanol) and with rifampicin (rifampin) as a positive control. mRNA levels were analysed using a branched DNA luminescent assay. CYP2B6 activity, protein and mRNA levels were increased by 2.5, 1.5 and 2.1 fold, respectively, by 20 microM rifampicin. However, 10 microM bupropion minimally altered CYP2B6 (1.4, 1.1, 0.8 fold). Although CYP3A4 activity, protein, and mRNA levels were increased by 4.0, 2.3, and 14.0 fold, respectively, by 20 microM rifampicin, 10 microM bupropion minimally altered CYP3A4 (1.4, 1.0, 0.8 fold). Rifampicin (20 microM) increased CYP2E1 protein by 2.1 fold, while 10 microM bupropion minimally altered CYP2E1 protein (1.2 fold). Overall, results of this study suggest that multiple doses of bupropion are not likely to induce CYP2B6, 3A4 or 2E1 in-vivo in man.

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“…A number of bDNA assays have been developed for quantification of viral nucleic acids, including human immunodeficiency virus type 1 (HIV-1) RNA (Kern et al 1996), simian immunodeficiency virus (SIV) RNA (Sodora et al 1998), hepatitis B virus (HBV) DNA (Hendricks et al 1995), hepatitis C virus (HCV) RNA , hepatitis G virus (HGV) RNA (Brandhagen et al 1999), and cytomegalovirus (CMV) DNA (Chernoff et al 1997;Pellegrin et al 2000). More recently, bDNA technology has been used to detect and measure the expression of cellular mRNAs, including cytokines (Breen et al 1997;Shen et al 1998), progesterone and estrogen receptors (Nargessi et al 1998a,b), insulin (Wang et al 1997), glucokinase (Cabrera-Valladares et al 1999), c-fos (Shyamala et al 1999), aP2 (Burris et al 1999), cytochrome P450 (Hartley and Klaassen 2000), and uncoupling proteins (Zhou et al 2000). Although these bDNA assays were developed to measure nucleic acids in serum, plasma, or cell lysates, other studies have shown that it is possible to adapt bDNA technology to an ISH format for detection of mRNA Antao et al 2000).…”

mentioning

confidence: 99%

Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization

Player

Shen

Kenny

et al. 2001

J Histochem Cytochem.

160

129

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We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

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A Branched DNA Signal Amplification Assay to Quantitate Messenger RNA of Human Uncoupling Proteins 1, 2, and 3

Cited by 15 publications

References 22 publications

Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists

Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists

Effect of bupropion on CYP2B6 and CYP3A4 catalytic activity, immunoreactive protein and mRNA levels in primary human hepatocytes: comparison with rifampicin

Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization

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