Open AccessJournal Article
PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR.
Siebert Pd,Larrick Jw +1 more
TLDR
It is shown that competitive PCR MIMICS can be used to accurately measure small changes in mRNA levels and is described as a rapid and reliable method for preparing competitive DNA fragments for quantitative PCR.Abstract:
A rapid and reliable method is described for preparing competitive DNA fragments for quantitative PCR. Synthetic DNAs complementary to previously established PCR primers are ligated together with the primers to both ends of a generic DNA fragment whose length differs from the natural target gene PCR product. After a short ligation step, the properly constructed ligation products (i.e., those that have the correct primer templates on opposite sides of the generic DNA fragment) are preferentially amplified by PCR. The generation of competitive PCR fragments, MIMICS, can be completed in a single day. To perform quantitative PCR, known quantities of PCR MIMICS are spiked into PCR amplification reactions containing the experimental cDNA samples. A visual or radioactive comparison of the PCR products can then be used to determine the initial quantity of target gene. We show that competitive PCR MIMICS can be used to accurately measure small changes in mRNA levels.read more
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Journal ArticleDOI
Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.
TL;DR: The technical aspects involved are discussed, conventional and kinetic RT-PCR methods for quantitating gene expression are contrasted, and the usefulness of these assays are illustrated by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
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B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4
Gordon J. Freeman,Vassiliki A. Boussiotis,Anukanth Anumanthan,Gregory M. Bernstein,Xiao Yen Ke,Paul D. Rennert,Gary S. Gray,John G. Grlbben,Lee M. Nadler +8 more
TL;DR: B7-1 and B7-2 costimulation mediate distinct outcomes, since B 7-2 provides an initial signal to induce naive T cells to become IL-4 producers, thereby directing the immune response more towards Th0/Th2, whereas B8-1 is a more neutral differentiative signal.
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Standardizing Chlamydia pneumoniae assays : Recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada)
Scott F. Dowell,Rosanna W. Peeling,Jens Boman,George M. Carlone,Barry S. Fields,Jeannette Guarner,Margaret R. Hammerschlag,Lisa A. Jackson,Cho-Chou Kuo,Matthias Maass,Trudy O. Messmer,Deborah F. Talkington,Maria Lucia Tondella,Sherif R. Zaki +13 more
TL;DR: Serological testing, culture, DNA amplification, and tissue diagnostics are reviewed, and standardized definitions for "acute infection" and "past exposure" are proposed, and the use of single immunoglobulin G titers for determining acute infection and IgA for determining chronic infection are discouraged.
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Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.
Lurdes Monteiro,Dominique Bonnemaison,Antoine Vekris,Klaus G. Petry,Jacques Bonnet,Rui Vidal,José Cabrita,Francis Mégraud +7 more
TL;DR: A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori, and these are complex polysaccharides possibly originating from vegetable material in the diet.
Journal ArticleDOI
Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT‐PCR)
M Niessner,B A Volk +1 more
TL;DR: The results demonstrate that chronic intestinal inflammation in patients with CD is characterized by an increase of Th1‐like cytokines, and suggests that IL‐10 is an important regulatory component involved in the control of the inflammatory response in inflammatory bowel disease.