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Topic:Real-Time PCR

Real-Time PCR (Polymerase Chain Reaction) is a molecular technique used to amplify and quantify DNA sequences in horses. This method allows for the detection and measurement of specific genetic material in real-time, providing valuable insights into genetic expression, pathogen presence, and disease diagnosis. In equine research, Real-Time PCR is utilized to study various aspects such as infectious diseases, genetic disorders, and gene expression profiles. The technique's sensitivity and specificity enable researchers to accurately assess the genetic material of interest, facilitating advancements in equine health diagnostics and management. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings related to Real-Time PCR in equine science.
Expression of bone morphogenetic protein-6 and -2 and a bone morphogenetic protein antagonist in horses with naturally acquired osteochondrosis.
American journal of veterinary research    January 15, 2004   Volume 65, Issue 1 110-115 doi: 10.2460/ajvr.2004.65.110
Semevolos SA, Nixon AJ, Strassheim ML.To determine the mRNA expression of bone morphogenetic protein (BMP)-6 and -2 and a BMP antagonist (Noggin) in horses with osteochondrosis. Methods: Samples of articular cartilage from affected stifle or shoulder joints of 10 immature horses with naturally acquired osteochondrosis and corresponding joints of 9 clinically normal horses of similar age; additionally, samples of distal femoral growth plate cartilage and distal femoral articular cartilage were obtained from a normal equine fetus. Methods: Cartilage specimens were snap-frozen in liquid nitrogen, and total RNA was isolated. Adjacent ...
Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation.
Veterinary immunology and immunopathology    October 3, 2003   Volume 96, Issue 1-2 31-41 doi: 10.1016/s0165-2427(03)00137-5
Fumuso E, Giguère S, Wade J, Rogan D, Videla-Dorna I, Bowden RA.Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial ...
Different CREM-isoform gene expression between equine and human normal and impaired spermatogenesis.
Theriogenology    September 27, 2003   Volume 60, Issue 7 1357-1369 doi: 10.1016/s0093-691x(03)00142-0
Blöcher S, Behr R, Weinbauer GF, Bergmann M, Steger K.Histone-to-protamine exchange causes chromatin condensation ceasing gene expression in elongating spermatids. Gene expression of protamines is regulated by the transcription factor cAMP-responsive element modulator (CREM). Altered CREM expression results in male infertility, as shown by CREM-knock-out mice being sterile due to round spermatid maturation arrest and patients exhibiting round spermatid maturation arrest revealing a lack or substantial reduction of both CREM-mRNA and CREM-protein. Similar defects in histone-to-protamine exchange have been suggested in infertile stallions exhibitin...
Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes.
Veterinary research    March 27, 2003   Volume 34, Issue 2 165-176 doi: 10.1051/vetres:2002063
Westcott DG, King DP, Drew TW, Nowotny N, Kindermann J, Hannant D, Belák S, Paton DJ.Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan assay,...
The effect of strenuous exercise on mRNA concentrations of interleukin-12, interferon-gamma and interleukin-4 in equine pulmonary and peripheral blood mononuclear cells.
Veterinary immunology and immunopathology    January 1, 2003   Volume 91, Issue 1 61-71 doi: 10.1016/s0165-2427(02)00274-x
Ainsworth DM, Appleton JA, Eicker SW, Luce R, Julia Flaminio M, Antczak DF.The effect of strenuous exercise on the mRNA concentrations of interleukin-12p35 subunit (IL-12p35), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in equine pulmonary and peripheral blood mononuclear cells (PBMCs) was investigated. We hypothesized that strenuous exercise would suppress the expression of IL-12p35, IFN-gamma and augment the expression of IL-4. Eleven horses were randomly divided into two groups, a stall-confined control group (n=5) and an exercise-conditioned treatment group (n=6). Bronchoalveolar and PBMCs were obtained from horses in the treatment group prior to the co...
Does training affect mRNA transciption for cytokine production in circulating leucocytes?
Equine veterinary journal. Supplement    October 31, 2002   Issue 34 154-158 doi: 10.1111/j.2042-3306.2002.tb05409.x
Colahan PT, Kollias-Bakert C, Leutenegger CM, Jones JH.Physical exertion is a stimulus for the upregulation of cytokine production including IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-10 and TNF-alpha in horses. To investigate that hypothesis, we initiated training of 5 stall-rested Thoroughbreds. Blood samples were drawn before and weekly during training. The relative transcription of mRNA within the leucocytes was measured using real time TaqMan quantitative PCR. The training protocol was walking (3 min), trotting (3 min) and cantering/galloping (6 min) increasing in intensity weekly (6 to 12 m/s) and culminating in an intense exercise period. Compa...
Detection of Salmonella spp in fecal specimens by use of real-time polymerase chain reaction assay.
American journal of veterinary research    September 13, 2002   Volume 63, Issue 9 1265-1268 doi: 10.2460/ajvr.2002.63.1265
Kurowski PB, Traub-Dargatz JL, Morley PS, Gentry-Weeks CR.To use real-time polymerase chain reaction (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. Methods: 299 fecal specimens from cattle, horses, and dogs. Methods: Enrichment of fecal specimens was followed by genomic DNA extraction by use of commercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. Results: Use of the spaQ pr...
Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV).
Journal of virological methods    August 15, 2002   Volume 105, Issue 1 171-179 doi: 10.1016/s0166-0934(02)00101-5
Cook RF, Cook SJ, Li FL, Montelaro RC, Issel CJ.A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6),...
Cytosolic ratio of malate dehyrogenase/lactate dehydrogenase activity in peripheral leukocytes of race horses with training.
Research in veterinary science    June 22, 2002   Volume 72, Issue 3 241-244 doi: 10.1053/rvsc.2002.0558
Arai T, Hosoya M, Nakamura M, Magoori E, Uematsu Y, Sako T.The activities of the enzymes involved in the malate-aspartate shuttle and m RNA expression of malate dehydrogenase (MDH), a crucial enzyme for the NADH shuttle that produces ATP in glucose metabolism in the peripheral leukocytes of horses, were measured to investigate the change in metabolic states with training. There were no significant differences in plasma glucose and immunoreactive insulin concentrations between race horses and riding horses, used as a comparable reference. The cytosolic and mitochondrial MDH activities in leukocytes of race horses were significantly higher than those of...
Expression and coassociation of ERG1, KCNQ1, and KCNE1 potassium channel proteins in horse heart.
American journal of physiology. Heart and circulatory physiology    June 14, 2002   Volume 283, Issue 1 H126-H138 doi: 10.1152/ajpheart.00622.2001
Finley MR, Li Y, Hua F, Lillich J, Mitchell KE, Ganta S, Gilmour RF, Freeman LC.In dogs and in humans, potassium channels formed by ether-a-go-go-related gene 1 protein ERG1 (KCNH2) and KCNQ1 alpha-subunits, in association with KCNE beta-subunits, play a role in normal repolarization and may contribute to abnormal repolarization associated with long QT syndrome (LQTS). The molecular basis of repolarization in horse heart is unknown, although horses exhibit common cardiac arrhythmias and may receive drugs that induce LQTS. In horse heart, we have used immunoblotting and immunostaining to demonstrate the expression of ERG1, KCNQ1, KCNE1, and KCNE3 proteins and RT-PCR to det...
Detection of equine arteritis virus by real-time TaqMan reverse transcription-PCR assay.
Journal of virological methods    February 19, 2002   Volume 101, Issue 1-2 21-28 doi: 10.1016/s0166-0934(01)00416-5
Balasuriya UB, Leutenegger CM, Topol JB, McCollum WH, Timoney PJ, MacLachlan NJ.A one-tube real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of equine arteritis virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time Ta...
Upregulation of mRNA of interleukin-1 and -6 in subchondral cystic lesions of four horses.
Equine veterinary journal    March 27, 2001   Volume 33, Issue 2 143-149 doi: 10.1111/j.2042-3306.2001.tb00592.x
von Rechenberg B, Leutenegger C, Zlinsky K, McIlwraith CW, Akens MK, Auer JA.This study investigated the potential association of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in subchondral cystic lesions (SCL) in horses. With the technique of in situ hybridisation in paraffin sections of fibrous tissue of SCL and quantitative real-time PCR in fresh frozen fibrous tissue and undecalcified bone sections of SCL embedded in acrylic resin, upregulation of mRNA of both cytokines could be demonstrated. mRNA of IL-1beta was upregulated at the periphery of the cystic lesion adjacent to normal bone, whereas IL-6 mRNA was upregulated within the fibrous tissue found with...
Characterization of cultured smooth muscle cells obtained from the palmar digital arteries of horses.
American journal of veterinary research    December 29, 2000   Volume 61, Issue 12 1602-1608 doi: 10.2460/ajvr.2000.61.1602
Rodgerson DH, Belknap JK, Fontaine GL, Kroll DL.To develop methods to isolate, culture, and characterize smooth muscle cells (SMC) from equine palmar digital arteries. Methods: Segments of the medial or lateral palmar digital arteries from the forelimbs of 6 horses. Methods: To obtain smooth muscle explants, arterial segments were incised longitudinally. The tunica intima was gently scraped from the underlying tunica media, and explants were obtained from the tunica media. Approximately 18 to 24 explants were obtained from each palmar digital arterial segment. A substrate-attached technique was used to initiate primary culture of SMCCulture...
Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR.
Veterinary parasitology    June 1, 2000   Volume 90, Issue 1-2 129-135 doi: 10.1016/s0304-4017(00)00227-2
Pusterla N, Leutenegger CM, Sigrist B, Chae JS, Lutz H, Madigan JE.A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evalua...
Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embedded in methyl methacrylate.
Calcified tissue international    December 14, 1999   Volume 65, Issue 5 378-383 doi: 10.1007/s002239900717
Leutenegger CM, von Rechenberg B, Huder JB, Zlinsky K, Mislin C, Akens MK, Auer J, Lutz H.Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and emb...
Quantitative evaluation of ehrlichial burden in horses after experimental transmission of human granulocytic Ehrlichia agent by intravenous inoculation with infected leukocytes and by infected ticks.
Journal of clinical microbiology    November 24, 1999   Volume 37, Issue 12 4042-4044 doi: 10.1128/JCM.37.12.4042-4044.1999
Pusterla N, Leutenegger CM, Chae JS, Lutz H, Kimsey RB, Dumler JS, Madigan JE.This paper describes the kinetics of the human granulocytic ehrlichiosis agent in the blood of horses experimentally infected by intravenous inoculation with infected leukocytes and by infected ticks as evaluated by using a real-time quantitative PCR assay. The data obtained indicated differences in the period of incubation, duration of rickettsemia, and initial and maximal ehrlichial loads between the two routes of infection.
[Detection of equine arteritis virus (EAV) in stallions–a contribution to the improvement of EAV diagnosis].
Berliner und Munchener tierarztliche Wochenschrift    February 24, 1999   Volume 112, Issue 1 10-13 
Starick E.Serum samples from 72 stallions were examined for the occurrence of antibodies against equine arteritis virus, of which 41 animals (57%) were found to be positive. 32 of the seropositive stallions were then screened for persistent EAV infection, before and after the breeding season. Semen samples were investigated by RT-PCR followed by dot blot hybridization and nested PCR, and by virus isolation on cell cultures as well. The carrier state was virologically confirmed in 11 of 32 stallions (34%) during the first and in 9 of 20 (45%) during the second investigation. RT-PCR followed by confirmato...
Quantitation of equine cytokine mRNA expression by reverse transcription-competitive polymerase chain reaction.
Veterinary immunology and immunopathology    February 9, 1999   Volume 67, Issue 1 1-15 doi: 10.1016/s0165-2427(98)00212-8
Giguère S, Prescott JF.A reverse transcription-competitive polymerase chain reaction (RT-cPCR) method was developed to quantitate equine interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p35, IL-12 p40, interferon-gamma (INF-gamma), tumor necrosis factor-alpha (TNF-alpha), and beta-actin mRNA expression. Using primers based on equine-specific sequences, these cytokines could be detected in concanavalin A-stimulated peripheral blood mononuclear cells. The specificity of the amplified product was confirmed by sequencing. For each cytokine, the assay was made quantitative by generating competitor ...
Endotoxin treatment of equine infectious anaemia virus-infected horse macrophage cultures decreases production of infectious virus.
The Journal of general virology    May 6, 1998   Volume 79 ( Pt 4) 747-755 doi: 10.1099/0022-1317-79-4-747
Smith TA, Davis E, Carpenter S.Lentiviruses replicate in cells of the immune system, and activation of immune cells has been shown to modulate virus replication. To determine the effects of macrophage activation on replication of equine infectious anaemia virus (EIAV), primary horse macrophage cultures (HMCs) were established from 20 different horses, infected with an avirulent strain of EIAV, and stimulated with 5 microg/ml of bacterial endotoxin. Supernatants collected from HMCs were assayed for the presence of tumour necrosis factor (TNF-alpha) and for production of infectious virus. Results indicated that EIAV replicati...
Molecular cloning of DNA for inhibin alpha-subunit from equine ovary.
The Journal of veterinary medical science    October 1, 1995   Volume 57, Issue 5 905-909 doi: 10.1292/jvms.57.905
Yamanouchi K, Yoshida S, Hasegawa T, Ikeda A, Chang KT, Matsuyama S, Nishihara M, Miyazawa K, Takahashi M.cDNA encoding equine inhibin alpha-subunit precursor protein was isolated from an equine ovarian cDNA library. For screening, the DNA probe was amplified by the RT-PCR using primers designed based on the rat inhibin alpha-subunit cDNA sequence. Out of 1.2 x 10(5) plaques screened, 19 positive clones were isolated, and one of these clones (Eq-alpha-11) contained a complete open reading frame encoding 367 amino acids. The similarity of the deduced amino acid sequences of both equine inhibin alpha-subunit precursor protein and the mature protein were greater than 80% to those of other six mammali...
[Development of PCR methods for detection of EAV infection].
   March 18, 2026  
The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen...
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