Abstract：
:The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibility of RT, PCR, RT-cPCR and measurement, amplifying the glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene from isolated renal glomeruli. We used an enzyme-linked immunosorbent assay (ELISA) to quantify PCR products. We also report our PCR-based method for constructing a competitor DNA identifiable independently of the native product. Our results show that the entire RT-PCR and ELISA process had a standard deviation (SD) of less than 10% (n = 10). This compared to an SD of less than 13% (n = 10) in PCR and ELISA. The SD for ELISA alone was less than 11% (n = 10). RT-cPCR quantitation gave an SD of approximately 15% (n = 10). These results support the use of standard RT-PCR for the relative quantitation of mRNA. RT-cPCR is also suited to relative quantitation, but it is also independent of the amplification saturation curve and permits the identification of differences in cellularity between samples.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Hall LL,Bicknell GR,Primrose L,Pringle JH,Shaw JA,Furness PNdoi
10.2144/98244rr02subject
Has Abstractpub_date
1998-04-01 00:00:00pages
652-8issue
4eissn
0736-6205issn
1940-9818journal_volume
24pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract：:A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1992-04-01 00:00:00
abstract：:In this study, we have developed an air-interface culture system in which guinea pig tracheobronchial epithelial (GPTE) cells rapidly form tight monolayers. Enzymatically isolated GPTE cells were plated on collagen-treated polycarbonate microporous cell culture inserts at a density of 10(6) cells/cm2 (day 0). Bioelect...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1993-09-01 00:00:00
abstract：:Extracellular vesicles (EVs) are spherical membrane structures released by most cells. These highly conserved mediators of intercellular communication carry proteins, lipids, and nucleic acids, and transfer these cellular components between cells by different mechanisms, such as endocytosis, macropinocytosis, or fusio...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114371
更新日期：2016-01-01 00:00:00
abstract：:A labeled peptide nucleic acid (PNA) antisense probe was used to study the spatial distribution of triplet repeats (CTG) in human myotonic dystrophy (DM) cells by high-resolution fluorescence in situ hybridization (FISH). It was found that transcripts containing triplet repeats were present as a number of discrete foc...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/98243rr02
更新日期：1998-03-01 00:00:00
abstract：:The expression of foreign proteins in Saccharomyces cerevisiae is a powerful tool for basic research and the biotechnological industry. In spite of the potential of S. cerevisiae, only a few useful expression vectors have been developed for this yeast. These vectors are based on an increasing transcription rate in com...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/00283rr04
更新日期：2000-03-01 00:00:00
abstract：:A method for the determination of prostaglandin G/H synthase and lipoxygenase activities in tissues was developed and employed with rat gastric mucosa samples. Tissues and microsomes were incubated in a buffer containing nonionic detergent and 1.32 mM arachidonic acid for 10 min. Following extraction with ethyl acetat...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-02-01 00:00:00
abstract：:A technique, Replacement PCR Mutagenesis, was developed to replace one immunoglobulin variable region (V) in a M13 phage cassette with a different, homologous V. This allows the use of the same mutagenesis and subsequent expression vectors for many V regions or V segments. The method combines PCR of V fragments and in...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1992-01-01 00:00:00
abstract：:Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selec...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114537
更新日期：2017-04-01 00:00:00
abstract：:Human embryonic kidney (HEK293) cells were stably transduced with a retroviral vector containing an expression cassette for a short-lived green fluorescent protein (d2EGFP) and the neomycin resistance gene (Neor). When Neor HEK293 clones were treated with proteasome inhibitors, lactacystin or MG132, an increase in the...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/01303dd03
更新日期：2001-03-01 00:00:00
abstract：:Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance c...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2019-0119
更新日期：2020-02-01 00:00:00
abstract：:Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as star...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/03343rr01
更新日期：2003-03-01 00:00:00
abstract：:Real-time PCR is becoming a preferred method for quantification of minute amounts of nucleic acids. To achieve the full potential of this technique, accurate and convenient models for post-PCR data analysis are required. In this study, three different models were chosen to quantify the definitive copy numbers of Cucum...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112750
更新日期：2008-06-01 00:00:00
abstract：:The serum protein transferrin (Tf) is a valuable marker for genetic studies of primates, because it is usually polymorphic, exhibiting as many as 13 allelic forms with high heterozygosity. The standard procedure to detect the different phenotypes requires vertical electrophoresis on polyacrylamide gels for 18 h at 4 d...
journal_title：BioTechniques
pub_type：
doi：
更新日期：1993-11-01 00:00:00
abstract：:Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR Green I, (ii) a decrease in fluorescein quenching by rhodamine after exonuclease cleavage of a dual-labeled hydrolysis probe and (iii) resonance ene...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/97221bi01
更新日期：1997-01-01 00:00:00
abstract：:A simple and rapid method for permanently marking autoradiographs is described. This procedure is based on the phosphorescence of light-activated zinc sulfide and the subsequent exposure of x-ray films by this light emission. A lacquer-based carrier allows the zinc sulfide to remain in suspension and permits permanent...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-05-01 00:00:00
abstract：:The technique described here is a fast and simple method of extracting chloroplast DNA (cpDNA). It overcomes the need for differential centrifugation using density gradients. The leaves do not have to be kept in the dark and lyophilized before extraction, but lyophilization is still possible. The chloroplasts are spec...
journal_title：BioTechniques
pub_type：
doi：10.2144/00281st07
更新日期：2000-01-01 00:00:00
abstract：:Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114377
更新日期：2016-02-01 00:00:00
abstract：:Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000113222
更新日期：2009-10-01 00:00:00
abstract：:Sequence-based methods for transcriptome characterization have typically relied on generation of either serial analysis of gene expression tags or expressed sequence tags. Although such approaches have the potential to enumerate transcripts by counting sequence tags derived from them, they typically do not robustly su...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112900
更新日期：2008-07-01 00:00:00
abstract：:Here, we present a rapid and reproducible procedure based on square-wave pulse electroporation that allows efficient penetration of synthetic oligonucleotides into intact yeast cells. This procedure was successfully used to modify the yeast genome with small amounts of oligonucleotide. ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/98252gt04
更新日期：1998-08-01 00:00:00
abstract：:Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, Eva...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114432
更新日期：2016-07-01 00:00:00
abstract：:Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112279
更新日期：2006-11-01 00:00:00
abstract：:Planar lipid bilayers represent a versatile platform for studying the functions of various membrane proteins as well as the development of biosensors. Despite the continuing technological progress in the fabrication of low-noise bilayer setups with mechanically and electrically stable planar bilayers, there is still a...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112384
更新日期：2007-03-01 00:00:00
abstract：:14CO2 capture is commonly used to evaluate the cellular oxidation rate of respiratory substrates. A modification of the established 14CO2-capture method was developed that enables the use of cells in adherent culture and easy analysis of multiple samples under different culture conditions. The use of commercially avai...
journal_title：BioTechniques
pub_type：
doi：10.2144/98245st04
更新日期：1998-05-01 00:00:00
abstract：:The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeri...
journal_title：BioTechniques
pub_type：
doi：10.2144/00292st04
更新日期：2000-08-01 00:00:00
abstract：:Cell-imaging approaches using new laser-based technologies have a wide applicability to thefields of pathology and cell biology. Here, we present the application of several of these techniques, including confocal scanning laser microscopy (CSLM), laser scanning cytometry (LSC), and laser capture microdissection (LCM),...
journal_title：BioTechniques
pub_type： 杂志文章,评审
doi：10.2144/01314rv01
更新日期：2001-10-01 00:00:00
abstract：:A direct method to study essential genes is to construct conditional knock-down mutants by replacement of their native promoter by an inducible one. In Mycobacteria, replacement of an essential gene promoter with an anhydrotetracycline inducible one was successfully used but required a multi-step approach. In this wor...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2018-0074
更新日期：2018-09-01 00:00:00
abstract：:This report describes a new method for labeling PCR-generated short length (60-120 bp) double-stranded DNA fragments for use as hybridization probes. The method utilizes gene-specific primers identical to those for PCR generation of non-radioactive DNA fragments. Radioactive probes are synthesized by Taq DNA polymeras...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1992-03-01 00:00:00
abstract：:Single-cell genomics (SCG) is a recently developed tool to study the genomes of unculturable bacterial species. SCG relies on multiple-strand displacement amplification (MDA), PCR, and next-generation sequencing (NGS); however, obtaining sufficient amounts of high-quality DNA from samples is a major challenge when per...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114389
更新日期：2016-03-01 00:00:00
abstract：:Using a simple electronic circuit, a thermocouple can be connected to a chart recorder to measure the actual temperature inside a PCR tube. This allows accurate inspection of the thermocycle program and comparison between thermoprofiles from different thermocyclers. We found that the recording of temperature cycling e...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-04-01 00:00:00
