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Real-time quantification of microRNAs by stem-loop RT-PCR
- Nucleic Acids Res
, 2005
"... A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related mi ..."
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A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
the review on
- D0 −D0 Mixing” in this Review
"... Abstract Recurrent spontaneous abortion (RSA) is de-fined as three or more consecutive pregnancy losses prior to the 20th week of gestation. The etiology of recurrent spontaneous abortion is often unclear and may be multifactorial, with much controversy regarding diag-nosis and treatment. Reasonably ..."
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Abstract Recurrent spontaneous abortion (RSA) is de-fined as three or more consecutive pregnancy losses prior to the 20th week of gestation. The etiology of recurrent spontaneous abortion is often unclear and may be multifactorial, with much controversy regarding diag-nosis and treatment. Reasonably accepted etiologic causes include, genetics, anatomical, endocrine, placen-tal anomalies, hormonal problems, infection, smoking and alcohol consumption, exposure to environmental factors, psychological trauma and stressful life event, certain coagulation and immunoregulatory protein de-fects. Detection of an abnormality in any of these areas may result into specific therapeutic measures, with varying degrees of success. However, the majority of cases of RSA remains unexplained and is found to be associated with certain autoimmune (APA, ANA, ACA, ATA, AECA) and alloimmune (APCA, Ab2, MLR-Bf) antibodies that may play major role in the immunologic failure of pregnancy and may lead to abortion. Alter-ation in the expression of HLA-G molecules, T-helper-1 (Th-1) pattern of cytokines and natural killer (NK) cells activity may also induce abortion. Various forms of treatment like antithrombotic therapies such as aspirin and heparin, intravenous immunoglobulin (IVIg) ther-apy, immunotherapy with paternal lymphocytes and vitamin D3 therapy are effective mode of treatment for unexplained cause of fetal loss in women with RSA.
Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes
- Cell
, 2008
"... Nuclear transcription is not restricted to genes but occurs throughout the intergenic and noncoding space of eukaryotic genomes. The functional significance of this widespread noncoding transcription is mostly unknown. We show that Arabidopsis RNA polymerase IVb/Pol V, a multisubunit nuclear enzyme ..."
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Cited by 43 (10 self)
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Nuclear transcription is not restricted to genes but occurs throughout the intergenic and noncoding space of eukaryotic genomes. The functional significance of this widespread noncoding transcription is mostly unknown. We show that Arabidopsis RNA polymerase IVb/Pol V, a multisubunit nuclear enzyme required for siRNA-mediated gene silencing of transposons and other repeats, transcribes intergenic and noncoding sequences, thereby facilitating heterochromatin formation and silencing of overlapping and adjacent genes. Pol IVb/Pol V transcription requires the chromatin-remodeling protein DRD1 but is independent of siRNA biogenesis. However, Pol IVb/Pol V transcription and siRNA production are both required to silence transposons, suggesting that Pol IVb/Pol V generates RNAs or chromatin structures that serve as scaffolds for siRNA-mediated heterochromatinforming complexes. Pol IVb/Pol V function provides a solution to a paradox of epigenetic control: the need for transcription in order to transcriptionally silence the same region.
Coordinate expression of fimbriae in uropathogenic Escherichia coli. Infect. Immun
, 2005
"... These include: This article cites 54 articles, 24 of which can be accessed free at: ..."
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Cited by 28 (11 self)
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These include: This article cites 54 articles, 24 of which can be accessed free at:
Fine-structure analysis of ribosomal protein gene transcription
- Mol. Cell. Biol
, 2006
"... These include: This article cites 42 articles, 18 of which can be accessed free at: ..."
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Cited by 28 (1 self)
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These include: This article cites 42 articles, 18 of which can be accessed free at:
MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators. Mol Cell Biol 28: 5369–5380
, 2008
"... Supplemental material This article cites 60 articles, 18 of which can be accessed free at: ..."
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Cited by 28 (0 self)
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Supplemental material This article cites 60 articles, 18 of which can be accessed free at:
Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana
- Plant Cell
, 2004
"... several weeks. ..."
Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli
- J
, 2006
"... Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5 � harboring pBR322, which is a common bacterial cloning vector. The se ..."
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Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5 � harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid �-lactamase gene (bla) and for the chromosomal d-1deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8–3.9% and 4.7–5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15–20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.
Eight-channel itraq enables comparison of the activity of 6 leukaemogenic tyrosine kinases
- Mol. Cell Proteomics
, 2007
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Chlamydial type III secretion system is encoded on ten operons preceded by sigma 70-like promoter elements
- J
, 2007
"... This article cites 64 articles, 36 of which can be accessed free ..."
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Cited by 17 (1 self)
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This article cites 64 articles, 36 of which can be accessed free
