A fluorescence-based assay for multisubunit DNA-dependent RNA polymerases.
AuthorsKuhlman P, Duff HL, Galant A
JournalAnal Biochem
PubMed ID14690681
The properties of DNA-dependent RNA polymerases have been studied since the 1960s, but considerable interest in probing RNA polymerase structure/function relationships, the roles of different classes of RNA polymerases in cellular processes, and the feasibility of using RNA polymerases as drug targets still exists. Historically, RNA polymerase activity has been ... More
Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR.
AuthorsMonpoeho S, Dehée A, Mignotte B, Schwartzbrod L, Marechal V, Nicolas JC, Billaudel S, Férré V
JournalBiotechniques
PubMed ID10907082
'We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the ... More
mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.
AuthorsScheidl SJ, Nilsson S, Kalén M, Hellström M, Takemoto M, Håkansson J, Lindahl P
JournalAm J Pathol
PubMed ID11891179
'Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of ... More
A mitotic cascade of NIMA family kinases. Nercc1/Nek9 activates the Nek6 and Nek7 kinases.
AuthorsBelham C, Roig J, Caldwell JA, Aoyama Y, Kemp BE, Comb M, Avruch J
JournalJ Biol Chem
PubMed ID12840024
'The Nek family of protein kinases in humans is composed of 11 members that share an amino-terminal catalytic domain related to NIMA, an Aspergillus kinase involved in the control of several aspects of mitosis, and divergent carboxyl-terminal tails of varying length. Nek6 (314AA) and Nek7 (303AA), 76% identical, have little ... More
Quantification of cDNA generated by reverse transcription of total RNA provides a simple alternative tool for quantitative RT-PCR normalization.
AuthorsLibus J, Storchová H
JournalBiotechniques
PubMed ID16925017
Multitasking with molecular dynamics Typhoon: quantifying nucleic acids and autoradiographs.
AuthorsGoodrich RJ, Ostermeier GC, Krawetz SA
JournalBiotechnol Lett
PubMed ID12889815
With increased sensitivity and specificity, fluorescent assays are rapidly becoming the method of choice for nucleic acid quantification. The utility of the Typhoon scanner has now been extended to accurately measure low levels of DNA and RNA (5 ng ml(-1)) with PicoGreen and RiboGreen dyes. In addition, with a few ... More
A sensitive and high-throughput assay to detect low-abundance proteins in serum.
AuthorsZhang H, Cheng X, Richter M, Greene MI
JournalNat Med
PubMed ID16532003
The ability to detect antigens immunologically is limited by the affinity of the antibodies and the amount of antigens. We have now succeeded in creating a modular, facile amplification system, termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). Such a system can detect protein targets specifically at subfemtomolar levels ... More
Gene expression analysis by transcript profiling coupled to a gene database query.
AuthorsShimkets RA, Lowe DG, Tai JT, Sehl P, Jin H, Yang R, Predki PF, Rothberg BE, Murtha MT, Roth ME, Shenoy SG, Windemuth A, Simpson JW, Simons JF, Daley MP, Gold SA, McKenna MP, Hillan K, Went GT, Rothberg JM
JournalNat Biotechnol
PubMed ID10429247
We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction ... More
MicroRNA-143 regulates adipocyte differentiation.
AuthorsEsau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun Y, Koo S, Perera RJ, Jain R, Dean NM, Freier SM, Bennett CF, Lollo B, Griffey R
JournalJ Biol Chem
PubMed ID15504739
MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) ... More
Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.
AuthorsRodgers JT, Patel P, Hennes JL, Bolognia SL, Mascotti DP
JournalAnal Biochem
PubMed ID10625515
We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin-agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect ... More
The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.
AuthorsBrown AJ, Goldsworthy SM, Barnes AA, Eilert MM, Tcheang L, Daniels D, Muir AI, Wigglesworth MJ, Kinghorn I, Fraser NJ, Pike NB, Strum JC, Steplewski KM, Murdock PR, Holder JC, Marshall FH, Szekeres PG, Wilson S, Ignar DM, Foord SM, Wise A, Dowell SJ
JournalJ Biol Chem
PubMed ID12496283
GPR41 and GPR43 are related members of a homologous family of orphan G protein-coupled receptors that are tandemly encoded at a single chromosomal locus in both humans and mice. We identified the acetate anion as an agonist of human GPR43 during routine ligand bank screening in yeast. This activity was ... More
RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization.
AuthorsJones LJ, Yue ST, Cheung CY, Singer VL
JournalAnal Biochem
PubMed ID9882416
We describe the development of a sensitive fluorescence-based solution assay for RNA using a new dye, RiboGreen RNA quantitation reagent. RiboGreen reagent exhibits >1000-fold fluorescence enhancement and high quantum yield (0.65) upon binding nucleic acids, with excitation and emission maxima near those of fluorescein. Unbound dye is essentially nonfluorescent and ... More
Binding of fluorescent dye to genomic RNA inside intact human rhinovirus after viral capsid penetration investigated by capillary electrophoresis.
AuthorsKremser L, Okun VM, Nicodemou A, Blaas D, Kenndler E
JournalAnal Chem
PubMed ID14961716
RiboGreen is used for concentration measurements of RNA. Upon binding to the RNA, an approximately 1000-fold increase in sensitivity in comparison with the UV absorbance of the free polynucleotide is observed. In the present work, we demonstrate that this dye can penetrate in a time- and temperature-dependent manner the intact ... More
Evaluation of internal standards in a competitive nucleic acid sequence-based amplification assay.
AuthorsLo WY, Baeumner AJ
JournalAnal Chem
PubMed ID17297938
An end-point quantitative nucleic acid sequence-based amplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyte E. coli clpB mRNA was developed and statistically analyzed. Electrochemiluminescence was chosen as a highly sensitive detection means allowing careful evaluation of the internal standards used. The two internal ... More
Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway.
AuthorsJadlowiec J, Koch H, Zhang X, Campbell PG, Seyedain M, Sfeir C
JournalJ Biol Chem
PubMed ID15371433
Extracellular matrix proteins (ECMs) serve as both a structural support for cells and a dynamic biochemical network that directs cellular activities. ECM proteins such as those of the SIBLING family (small integrin-binding ligand glycoprotein) could possess inherent growth factor activity. In this study, we demonstrate that exon 5 of dentin ... More
Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins.
AuthorsUlbrich P, Haubova S, Nermut MV, Hunter E, Rumlova M, Ruml T
JournalJ Virol
PubMed ID16809314
In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion ... More
RNA internal standard synthesis by nucleic acid sequence-based amplification for competitive quantitative amplification reactions.
AuthorsLo WY, Baeumner AJ
JournalAnal Chem
PubMed ID17297954
Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesize new sequences based on deletion and insertion reactions. Two RNA internal standards were synthesized for use in competitive amplification reactions in which quantitative analysis can be achieved by coamplifying the internal standard with the wild type sample. The ... More
Maturation of White Adipose Tissue Function in C57BL/6j Mice From Weaning to Young Adulthood.
AuthorsKodde A, Engels E, Oosting A, van Limpt K, van der Beek EM, Keijer J
JournalFront Physiol
PubMed ID31354508
White adipose tissue (WAT) distribution and WAT mitochondrial function contribute to total body metabolic health throughout life. Nutritional interventions starting in the postweaning period may impact later life WAT health and function. We therefore assessed changes in mitochondrial density and function markers in WAT depots of young mice. Inguinal (ING), ... More