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1.
Sergei V. Saveliev 《Biological procedures online》2002,4(1):70-80
The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit
of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA
before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional
PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions
in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications
at the ends of transfected DNA during gene therapy trials.
Published: November 11, 2002 相似文献
2.
We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells
retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected
to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s
worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered
commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details
and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure
preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general.
Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific
study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this
approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years
ahead. 相似文献
3.
Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from
microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing
to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays
and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (ρ=0.708, p<0.0001,
n=277) using these platforms. The contribution of factors including up — vs. down-regulation, spot intensity, ρ-value, fold-change,
cycle threshold (Ct), array averaging, tissue type, and tissue preparation was assessed. Filtering of microarray data for measures of quality
(fold-change and ρ-value) proves to be the most critical factor, with significant correlations of ρτ;0.80 consistently observed
when quality scores are applied. 相似文献
4.
Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric
cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white
blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We
report methodology using primers for a portion of the H2-kb murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000
μM primer, a 20-fold increase in the median manufacturer’s recommended concentration, the assay could be optimized to detect
34 pg of C57BL/6J DNA in a background of 2.5 μg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection
limit half as sensitive as that found when no carrier DNA was present.
Published: April 7, 2003 相似文献
5.
We have developed a simple and effective method (Lig-PCR) for monitoring ligation reactions using PCR and primers that are
common to many cloning vectors. Ligation mixtures can directly be used as templates and the results can be analyzed by conventional
gel electrophoresis. The PCR products are representative of the recombinant molecules created during ligation and the corresponding
transformants. Orientation of inserts can also be determined using an internal primer. The usefulness of this method has been
demonstrated using ligation mixtures of two cDNA’s derived from the salivary glands of Aedes aegypti mosquitoes. The method described here is sensitive and easy to perform compared to currently available methods. 相似文献
6.
Techniques of EMG signal analysis: detection, processing, classification and applications 总被引:1,自引:0,他引:1
Electromyography (EMG) signals can be used for clinical/biomedical applications, Evolvable Hardware Chip (EHW) development,
and modern human computer interaction. EMG signals acquired from muscles require advanced methods for detection, decomposition,
processing, and classification. The purpose of this paper is to illustrate the various methodologies and algorithms for EMG
signal analysis to provide efficient and effective ways of understanding the signal and its nature. We further point up some
of the hardware implementations using EMG focusing on applications related to prosthetic hand control, grasp recognition,
and human computer interaction. A comparison study is also given to show performance of various EMG signal analysis methods.
This paper provides researchers a good understanding of EMG signal and its analysis procedures. This knowledge will help them
develop more powerful, flexible, and efficient applications. 相似文献
7.
8.
Real-time polymerase chain reaction (PCR) constitutes a significant improvement over traditional end-point PCR, as it allows
the quantification of starting amounts of nucleic acid templates, in real-time. However, quantification requires validation
through numerous internal controls and standard curves. We describe in this paper a simple protocol which uses real-time PCR
to compare mRNA levels of a gene of interest between different experimental conditions. Comparative real-time PCR can be a
relatively low-cost method and does not require sequence-specific fluorescent reporters. Moreover, several genes from a set
of experiments can be assessed in a single run. Thus, in addition to providing a comparative profile for the expression of
a gene of interest, this method can also provide information regarding the relative abundance of different mRNA species. 相似文献
9.
We developed a rapid mutagenesis method based on a modification of the QuikChange® system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 – 22 bp to achieve melting temperatures greater than 80°C. 2: the final concentrations of both primers were increased to 5–10 ng/µl and the final concentration of template to 1–2 ng/µl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest. 相似文献
10.
In platelets, PGHS-1-dependant formation of thromboxane A2 is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin.
Since platelets are anucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01, which can be
induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression.
Using a specific antibody to PGHS-1 we have developed a technique using immunofluorescence microscopy and analysis of multiple
digital images to monitor PGHS-1 protein expression as MEG-01 cells were induced to differentiate by a single addition of
TPA (1.6 × 10−8 M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique
we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised
viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen,
CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation
between PGHS-1 expression and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein expression,
which shows a consistent increase over the entire course of differentiation can be used as an additional and better index
by which to monitor megakaryocyte differentiation.
Published: December 12, 2001 相似文献
11.
It is widely acknowledged that the presence of extracellular matrix components as substrates can drastically modulate the
phenotype and gene expression of cultured cells, including tumor cells. A number of published reports indicated that substrates
made from two peculiar collagen species, i.e. type V and OF/LB, which are abnormally deposited in the stroma of primary ductal
infiltrating carcinoma (d.i.c.) of the breast “in vivo,” were able to exert marked and opposite effects on “in vitro” viability,
growth and invasiveness of the 8701-BC cell line, isolated from d.i.c.-affected breast epithelium. To complement such functional
data on the effect of cell-collagen interactions with information at molecular level, we have utilized a combination of differential
display- and semi-quantitative multiplex-PCR techniques with the aim of detecting variations in the expression levels of selected
genes by cells maintained in either culture condition. Here we report some prototypical data on the identification and semi-quantitation
of three of the differentially-amplified PCR products found, i.e.HSP2A andMSF-B which are up-regulated in cells grown onto OF/LB collagen substrate, andSRCAP which is prominently down-regulated in the presence of type V collagen substrate. This protocol represents a powerful tool
for evaluating changes in the levels and patterns of gene expression which can be theoretically adapted to any experimental
model system.
Published: November 24, 2003 相似文献
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Due to the involvement of calcium as a main second messenger in the plant signaling pathway, increasing interest has been
focused on the calcium signatures supposed to be involved in the patterning of the specific response associated to a given
stimulus. In order to follow these signatures we described here the practical approach to use the non-invasive method based
on the aequorin technology. Besides reviewing the advantages and disadvantages of this method we report on results showing
the usefulness of aequorin to study the calcium response to biotic (elicitors) and abiotic stimuli (osmotic shocks) in various
compartments of plant cells such as cytosol and nucleus.
Published: December 9, 2002 相似文献
15.
Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a
short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member
Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects
on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice
to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone. 相似文献
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17.
In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100–150 mM. It was recently discovered
that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI) with 9–11 predicted transmembrane spanning
domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12
cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five
rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack
of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes.
The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake
in vitro using lipid-detergent vesicles.
Published: June 7, 2004. 相似文献
18.
An improved method for constructing and selectively silanizing double-barreled,neutral liquid-carrier,ion-selective microelectrodes 总被引:1,自引:0,他引:1
We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective
microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs
a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several
multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less
handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and
employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for
up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore
in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ∼150 μm through the abaxial surface. Microelectrode readings remained stable
after multiple impalements without the need for a stabilizing PVC matrix. 相似文献
19.
Matthew E. Bechard Sonya Chhatwal Rosemarie E. Garcia Madeline E. Rasche 《Biological procedures online》2003,5(1):69-77
Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria.
The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways
of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5′-phosphate synthase (RFAP synthase). Given the importance of
RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide
an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes
has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies inEscherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing
recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression,
RFAP synthase fromArchaeoglobus fulgidus was produced inE. coli and purified to homogeneity. The production of active RFAP synthase fromMethanothermobacter thermautotrophicus was achieved by coexpression of the geneMTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active
RFAP synthase.
Florida Agricultural Experiment Station Journal Series no. R-09353
Published: March 4, 2003 相似文献