Visualization of Gene Expression by Fluorescent Multiplex Reverse Transcriptase-PCR Amplification

2007 ◽  
pp. 143-152
Author(s):  
María Rosa Ponce ◽  
Víctor Quesada ◽  
Andrea Hricová ◽  
José Luis Micol
Keyword(s):  
Gene Expression ◽  
Reverse Transcriptase ◽  
Pcr Amplification ◽  
Reverse Transcriptase Pcr

Comparison of storage methods for reverse-transcriptase PCR amplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecal samples

2004 ◽  
Vol 116 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Christopher A Whittier ◽  
William Horne ◽  
Barrett Slenning ◽  
Michael Loomis ◽  
Michael K Stoskopf
Keyword(s):  
Reverse Transcriptase ◽  
Pcr Amplification ◽  
Gorilla Gorilla ◽  
Fecal Samples ◽  
Reverse Transcriptase Pcr ◽  
Storage Methods

scholarly journals Reverse Transcriptase-PCR Analysis of Bacterial rRNA for Detection and Characterization of Bacterial Species in Arthritis Synovial Tissue

2000 ◽  
Vol 68 (10) ◽  
pp. 6012-6026 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Michael Hurle ◽  
Anthony Wong ◽  
Scott Wilkie ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Synovial Tissue ◽  
Bacterial Species ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Modified Technique ◽  
Culture Methods ◽  
Reverse Transcriptase Pcr

ABSTRACT Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.

Rotavirus G2P[4] Detection in Fresh Vegetables and Oysters in Mexico City

2014 ◽  
Vol 77 (11) ◽  
pp. 1953-1959 ◽  
Author(s):  
CAROLINA QUIROZ-SANTIAGO ◽  
CARLOS VÁZQUEZ-SALINAS ◽  
IVAN NATIVIDAD-BONIFACIO ◽  
BLANCA LILIA BARRÓN-ROMERO ◽  
ELSA IRMA QUIÑONES-RAMÍREZ
Keyword(s):  
Reverse Transcriptase ◽  
Viral Infections ◽  
Pcr Amplification ◽  
Detection Sensitivity ◽  
Gastrointestinal Infections ◽  
Reverse Transcriptase Pcr ◽  
Vegetable Samples ◽  
Fresh Vegetables ◽  
Contaminated Food ◽  
Combined Genotypes

Rotaviruses are the principal cause of dehydration caused by diarrhea in children younger than 2 years of age. Although these viral infections have mainly been associated with ingestion of fecally contaminated food and water, few studies have addressed the presence of the virus in food that is consumed raw or slightly cooked. In this work, 30 oyster samples and 33 vegetable samples were examined for the presence of rotavirus genotypes to evaluate their potential to produce gastrointestinal infections. The rotaviruses were identified by reverse transcriptase PCR amplification of the VP7 gene. G and P genotyping was also performed by reverse transcriptase PCR, with a detection sensitivity of up to 15 PFU/ml. Rotaviruses were found in 17 (26.9%) of 63 samples (10 oysters and 7 vegetables). The G2 genotype was found in 11 (64.7%) of 17 of the rotavirus strains, and 16 (94.1%) of 17 had the P[4] genotype. The combined genotypes found most frequently were G2P[4] (10 [58.82%] of 17), GNTP[4] (6 [35.29%] of 17), and G2P[NT] (1 [5.8%] of 17).

Assessment of Environmental Factors on Listeria monocytogenes Scott A inlA Gene Expression by Relative Quantitative Taqman Real-Time Reverse Transcriptase PCR

2006 ◽  
Vol 69 (11) ◽  
pp. 2754-2757 ◽  
Author(s):  
SCOTT E. HANNA ◽  
HUA H. WANG
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Environmental Factors ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Internal Standard ◽  
Reverse Transcriptase Pcr ◽  
The Impact

Several virulence factors are involved in Listeria monocytogenes pathogenicity. L. monocytogenes internalins, particularly internalin A, are required for bacterial adhesion to and invasion of human intestinal epithelial cells. The expression of inter-nalins is thus related to virulence. Identification of conditions involved in regulating the expression of L. monocytogenes virulence factors is essential for developing targeted strategies to control listeriosis incidence and improving therapeutic approaches. The primary aim of this study was to develop a quantitative real-time reverse transcriptase PCR platform to study the impact of environmental factors on L. monocytogenes Scott A virulence factor expression, particularly in potentially complex ecosystems. A Taqman PCR–based, rapid quantitative gene expression evaluation method was established with the L. monocytogenes ribosomal protein L4 encoding gene used as an internal standard. Our data suggest that inlA expression is influenced by food composition and temperature, indicating that certain food processing or storage conditions, such as the use of lactic and acetic acids at common storage temperatures, could affect the expression of L. monocytogenes virulence factor.

scholarly journals Poly(A) Polymerase Modification and Reverse Transcriptase PCR Amplification of Environmental RNA

2005 ◽  
Vol 71 (3) ◽  
pp. 1267-1275 ◽  
Author(s):  
Lina M. Botero ◽  
Seth D'Imperio ◽  
Mark Burr ◽  
Timothy R. McDermott ◽  
Mark Young ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Cdna Library ◽  
Cdna Clone ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Random Priming ◽  
Rna Targeting ◽  
Pure Cultures ◽  
Heated Soil

ABSTRACT We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the α-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.

scholarly journals Assessment of Helicobacter pylori Gene Expression within Mouse and Human Gastric Mucosae by Real-Time Reverse Transcriptase PCR

2001 ◽  
Vol 69 (8) ◽  
pp. 4759-4766 ◽  
Author(s):  
Bachra Rokbi ◽  
Delphine Seguin ◽  
Bruno Guy ◽  
Véronique Mazarin ◽  
Emmanuel Vidor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
16S Rrna ◽  
Mouse Model ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
H Pylori

ABSTRACT Despite increasing knowledge on the biology of Helicobacter pylori, little is known about the expression pattern of its genome during infection. While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed. In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa. The expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. We found that the selected genes were all expressed within both mouse and human infected mucosae. Moreover, the relative abundance of transcripts was the same (16S rRNA > ureA >katA > alpA), in the two models. Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (P < 0.05 for 16S rRNA, alpA, andkatA). Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification ofH. pylori gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with H. pylori provide a valuable model for the analysis of bacterial gene expression during infection.

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