Quantitative Competitive Reverse Transcriptase-polymerase Research Articles

Development of Quantitative Competitive—Reverse Transcriptase—Polymerase Chain Reaction for Detection and Quantitation of Avian Leukosis Virus Subgroup J

Infection with avian leukosis virus subgroup J (ALV-J) causes severe economic losses in the broiler industry by increasing mortality, producing tumors, and decreasing weight gain in chickens. The quantitation of ALV-J is difficult because of its failure to produce a cytopathic effect in cell culture systems and the nonspecificity of antigen-capture enzyme-linked immunosorbent assay (ELISA) tests. This study was performed to develop a quantitative competitive-reverse transcriptase-polymerase chain reaction (QC-RT-PCR) method based on coamplification of ALV-J genomic RNA and a known amount of a synthesized RNA competitor. The 369 bp RNA competitor was constructed by restriction enzyme treatment of an ALV-J specific 545 bp PCR product, ligation, transformation into Escherichia coli, and in vitro transcription. The competitor contained the same amplification primer annealing sites and sequence as the original viral RNA, except that it had a 176 bp internal deletion. Coamplified RT-PCR products were visualized by electrophoresis and ethidium bromide staining, and fluorescences were quantified using computer-assisted image analysis. The sensitivity of this new QC-RT-PCR method was 25 fg of viral RNA, and 10-fold dilutions were differentiable. This method allowed absolute and relative quantification of ALV-J RNA copy numbers and was simpler than previously published methods for ALV-J quantification.

GABA A α 1 and α 2 receptor subunit expression in rostral ventrolateral medulla in nonpregnant and pregnant rats

Pregnancy results in attenuated baroreflex mediated sympathoexcitatory responses which may be due to potentiation of γ-aminobutyric acid (GABA) inhibition in the rostral ventrolateral medulla (RVLM). The major metabolite of progesterone, 3α-hydroxy-dihydroprogesterone (3α-OH-DHP), which is elevated in pregnancy, is a potent neurosteroid positive modulator of GABA A receptors, and sensitivity of GABA A receptors to 3α-OH-DHP is dependent on the receptor subunit composition. The purpose of this study was to evaluate the GABA A α 1 and α 2 receptor subunit mRNA and protein expression in the RVLM of nonpregnant and late term pregnant rats. Micropunches of RVLM were collected from nonpregnant and late term pregnant rats and the expression levels of GABA A α 1 and α 2 receptor subunits were analyzed using quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot techniques. The competitive RT-PCR analysis allows comparison of expression levels between different mRNA, and the mRNA expression level of GABA A α 1 was several hundred fold greater than GABA A α 2 in both groups. However, this relative distribution of GABA A α 1 and α 2 receptor subunits protein or mRNA expression was not altered in late term pregnant compared to nonpregnant rats. These data demonstrate, that within the RVLM of both nonpregnant and late term pregnant rats, the relative expression levels of GABA A α 1,2 receptor subunits favor GABA A receptors susceptible to positive modulation by progesterone metabolites.

Blood and intragraft CD27 gene expression in cardiac transplant recipients

The present study investigated gene expression of costimulatory molecule CD27 in relation to the occurrence of acute cardiac rejection. CD27 transcripts were measured by means of quantitative competitive reverse transcriptase–polymerase chain reaction in 120 endomyocardial biopsies and in 89 samples of blood mononuclear cells from 31 recipients. Higher levels of CD27 transcripts were observed in biopsies with rejection than in samples without rejection (medians, 7.1 and 1.9; P = 0.06). In contrast, blood mononuclear cells collected during rejection showed lower levels than blood mononuclear cells from rejection-free periods (medians, 3.3 vs. 7.9; P = 0.03). Considering only endomyocardial biopsies without rejection, the values were lower in samples from recipients who did not present any rejection during the first 6 months after transplantation than in those from recipients who had at least one rejection during the same period (medians, 0 vs. 3.5, P < 0.001; percentage of biopsies expressing CD27, 44% vs. 77%). In conclusion, the presence of intragraft CD27 mRNA may identify recipients at risk for developing acute rejection.

Expression of granzyme B mRNA is altered in human immunodeficiency virus infected patients

CD8-positive cytotoxic T lymphocytes and natural killer cells are the major cytotoxic components of the antiviral immune response. The major pathway used by these cells in response to viral-infected cells involves granzymes, cytotoxic granule serine proteases involved in the pathway leading to target cell DNA fragmentation and apoptosis. The levels of granzyme B mRNA in peripheral blood cells of human immunodeficiency virus (HIV) type-1 infected patients in comparison to noninfected individuals were assessed by quantitative competitive reverse transcriptase polymerase chain reaction. Expression of granzyme B mRNA is altered in HIV-1 infected patients. Significantly fewer HIV patients had detectable granzyme B mRNA levels than controls. The one HIV-infected patient with detectable granzyme B mRNA displayed a much higher level of this mRNA than all healthy controls. Cell-mediated cytotoxicity during HIV-1 infection may be impaired due to a deficient quantity of active cytotoxic granules or to their abnormal regulation.

Development of a quantitative competitive reverse transcriptase polymerase chain reaction for the quantification of growth hormone gene expression in pigs

After the advent of the genome projects, followed by the discovery of DNA polymorphisms, basic understanding of gene expression is the next focus to explain the association between polymorphisms and the level of gene expression, as well as to demonstrate the interaction among genes. Among the various techniques for the investigation of transcriptional profiling involving patterns of gene expression, quantitative PCR is the simplest analytical laboratory technique. The objective of this work was to analyze two strategies of a competitive PCR technique for the quantification of the pig growth hormone (GH) gene expression. A pair of primers was designed targeting exons 3 and 5, and two competitive PCR strategies were performed, one utilizing a specific amplicon as a competitor, and the other utilizing a low-stringency PCR amplicon as a competitor. The latter strategy proved to be easier and more efficient, offering an accessible tool that can be used in any kind of competitive reaction, facilitating the study of gene expression patterns for both genetics and diagnostics of infectious diseases.

Expression of allatostatin in the oviducts of the cockroach Diploptera punctata

The cockroach allatostatins (Y/FXFGL/Ia ASTs) are a ubiquitous family of peptides in the invertebrates. They affect numerous physiological processes including the inhibition of juvenile hormone III (JH) biosynthesis, inhibition of muscle contraction, inhibition of ovarian ecdysteroid biosynthesis and inhibition of vitellogenin (Vg) release from the fat body. We have developed and optimized a sensitive and specific quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR) method to quantify Diploptera punctata AST (Dippu-AST) expression. Using this technique we show that tissues of both lateral and common oviducts and the ovary express message for Dippu-AST. Moreover, the pattern of expression observed in the oviducts and ovary is strikingly similar with significant changes occurring during the reproductive cycle. Specifically, expression of AST is drastically reduced during the time of maximal vitellogenin (Vg) uptake, with higher levels measured prior to and following vitellogenesis. Furthermore, using immunocytochemistry, we have shown Dippu-AST-like-immunoreactivity in the terminal abdominal ganglion, as well as in ventral nerve 7, some branches of which innervate the common and lateral oviducts with other branches innervating the bursa copulatrix and brood sac of mated female D. punctata. The pattern of Dippu-AST expression and immunocytochemical staining suggests that ASTs function, in part, to regulate the cycle of vitellogenesis in mated female D. punctata.

Quantitation of liver-stage parasites by competitive RT-PCR.

A standard method for evaluating inhibitory effects on preerythrocytic parasites in murine malaria models is to measure the time to patency of a blood-stage infection following a sporozoite challenge. The prevention of a blood-stage infection or a delay in patency suggests that preerythrocytic parasites have been eliminated or that liverstage development has been inhibited. However, susceptibility to a challenge with blood-stage parasites must also be determined to conclude that preerythrocytic parasites and not blood-stage parasites were targeted. As a more direct method, liver-parasite burden can be measured. Due to the paucity of liver-stage parasites at physiological challenge doses, 1.0 × 106 sporozoites) (2). Molecular techniques that target Plasmodium-specific small subunit rRNA are much more sensitive, requiring less than 5.0 × 104 sporozoites to quantitate liver stages (3,4). Plasmodium rRNA has been now measured in total liver RNA by spot-blot hybridization with Plasmodium-specific rRNA probes (3) and as described here by quantitativecompetitive reverse transcriptase–polymerase chain reaction (RT-PCR) (4).

Interleukin-11 Attenuates Nephrotoxic Nephritis in Wistar Kyoto Rats

Interleukin-11 (IL-11) is a multifuctional cytokine with anti-inflammatory activity. The effect of IL-11 was studied in an experimental model of necrotizing glomerulonephritis induced in Wistar Kyoto rats by an injection of anti-glomerular basement membrane antibody (nephrotoxic serum). Intraperitoneal injection was chosen as the route of IL-11 administration in all experiments. In experiment 1, recombinant human IL-11 (1360 microg) was given 2 h before nephrotoxic serum, then once daily until day 6. In experiment 2, a lower dose of IL-11 (800 microg/d) was used. Rats were treated either with IL-11 400 microg twice daily intraperitoneally or with 800 microg once daily intraperitoneally for 6 d. In experiment 3, the lower dose of IL-11 was given 2 h before nephrotoxic serum, then twice daily until day 2. In experiment 1, IL-11 significantly reduced proteinuria (13.2 +/- 3.3 versus 63.2 +/- 4.3 mg/24 h), fibrinoid necrosis (0.58 +/- 0.08 versus 1.52 +/- 0.06 quadrants/glomerular cross section [gcs]), macrophage infiltration (ED1-positive cells, 24.4 +/- 1.8 versus 39.3 +/- 1.9 cells/gcs), apoptosis (1.11 +/- 0.1 versus 2.39 +/- 0.2 apoptotic bodies/gcs), and proliferating cell nuclear antigen-positive cells (24.4 +/- 2.0 versus 37.3 +/- 2.3 cells/gcs). Inducible nitric oxide synthase-positive cells were significantly increased (3.1 +/- 0.3 versus 2.0 +/- 0.2 cells/gcs). In experiment 2, a lower dose of IL-11 significantly reduced proteinuria and fibrinoid necrosis. Macrophage infiltration was similar in treated and control groups, although the number of sialoadhesin-positive macrophages (ED3+) was significantly reduced in the IL-11-treated rats. In experiment 3, quantitative competitive reverse transcriptase-polymerase chain reaction showed that the mRNA ratio of IL-1 beta/beta-actin in the treated rats was reduced compared with controls. By the use of probes designed from mouse IL-11 receptor alpha-chain sequence, it was also shown that rat mesangial cells and macrophages expressed IL-11 receptor alpha-chain, demonstrating that they were capable of responding to IL-11. In this model of necrotizing glomerulonephritis, high-dose IL-11 treatment markedly reduced both proteinuria and fibrinoid necrosis. At the lower dose, there was a reduction in glomerular injury and macrophage sialoadhesin expression, but without an alteration of macrophage numbers, suggesting that IL-11 may be acting in part to reduce macrophage activation.

Human immunodeficiency virus type 2 (HIV-2) in Portugal: Clinical spectrum, circulating subtypes, virus isolation, and plasma viral load

The human immunodeficiency virus type 2 (HIV-2) is responsible for 4. 5% of AIDS cases in Portugal. Six HIV-2 subtypes have been described so far, subtype A being proposed as more pathogenic than the rest. The relationship between the clinical status and levels of both cellular and plasma HIV-2 viraemia is not well known, nor their modifications under antiretroviral therapy. Thirty-two consecutive HIV-2 infected persons (17 men, 15 women) attending two different hospitals in Lisbon in 1997 were enrolled prospectively in the study. All but 4 individuals most likely acquired the infection through heterosexual contact. More than half of the study population was of African origin, mainly from Guinea-Bissau. Eleven (34.4%) patients had developed clinical manifestations included within the B or C groups of the CDC classification system for HIV infection, with the rest being asymptomatic. Half of the population was undergoing antiretroviral treatment at the time of the study. HIV-2 subtypes were investigated using a new Nef-based restriction fragment length polymorphism (RFLP) method that allows differentiation of the main two variants, A and B. Plasma viral load was quantified using a new quantitative competitive reverse transcriptase polymerase chain reaction (QcRT-PCR) procedure as well as the Amp-RT assay. Virus isolation was attempted from peripheral blood mononuclear cells. All but one person carried HIV-2 subtype A. Plasma viraemia examined by QcRT-PCR was measurable in 15 (50%) of 30 subjects, yielding in all instances values below 20,000 HIV-2 RNA copies per ml. Plasma RT activity could be detected in only 10 (33%) of 30 subjects, a rate much lower than that seen in HIV-1 infection. Virus was isolated from 16 (53.3%) of 30 patients. A significant correlation was found between CD4+ counts, clinical status, rate of virus isolation, and plasma viral load by both QcRT-PCR and Amp-RT. In conclusion, HIV-2 subtype A is the predominant variant circulating in Portugal among both natives and immigrants. A lower cellular and plasma viral load with respect to HIV-1 was seen in persons without immunosuppression, from whom the rate of virus recovery was extremely low.

Patterns of CYP26 expression in human prenatal cephalic and hepatic tissues indicate an important role during early brain development

CYP26 ( P450RAI) catalyzes catabolic retinoic acid (RA) hydroxylation and thereby appears to play a critical role in retinoid signaling pathways during development. In this study, a quantitative competitive reverse transcriptase–polymerase chain reaction (RT-PCR) assay was developed for evaluation of CYP26 message levels in human prenatal tissues. Statistical analyses of transcription levels in 12 prenatal human brains and six prenatal human livers demonstrated good sensitivity and reproducibility. Quantitative profiles of CYP26 gene expression in early (gestational days 57–110) prenatal cephalic and hepatic tissues and comparisons with adult counterparts are reported for the first time. Prenatal cephalic tissues at days 57–67 exhibited values of 1950±420 (CYP26 molecules/10 6 GAPDH molecules) whereas prenatal cephalic tissues at days 105–110 exhibited values of 22 300±4450 (CYP26 molecules/10 6 GAPDH molecules), indicating a sharp developmental increase (approximately 11-fold). Levels in human adult cephalic tissues were slightly less than the prenatal cephalic levels measured during the earliest stages of gestation and were approximately 3-fold lower than those measured in adult human hepatic tissues. Levels in human prenatal hepatic tissues at days 63–110 gestation were less than 800 (CYP26 molecules/10 6 GAPDH molecules) and did not exhibit developmental increases. Considered together, the data have strong implications for the importance of CYP26 in early development of the human brain.

Viremia and AIDS in rhesus macaques after intramuscular inoculation of plasmid DNA encoding full-length SIVmac239.

We have succeeded in stably maintaining the entire genome of SIVmac239 as a plasmid clone. Supercoiled proviral plasmid DNA was inoculated intramuscularly into two adult rhesus macaques and into a neonate. All three animals became viremic and seroconverted. Viral kinetics were followed prospectively by quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR), measurement of proviral DNA load in peripheral blood mononuclear cells (PBMCs) by PCR, and virus isolation by cocultivation. The infant developed high virus loads and succumbed to AIDS and SIV-associated nephropathy at 10 weeks postinoculation. Both adults are still living but have progressed to AIDS; one adult has also developed severe thrombocytopenia. We conclude that infection through intramuscular inoculation of cloned plasmid DNA encoding the entire proviral genome is reproducible and will provide a useful tool for studying viral pathogenesis.

Simultaneous Measurement of Multiple mRNAs with a Single Control by Quantitative Competitive Reverse Transcriptase–Polymerase Chain Reaction: Glucose Transporters Glut1 and Glut4

Theoretical considerations for extending the application of quantitative competitive polymerase chain reaction (qc-PCR) to include the simultaneous measurement of multiple mRNAs, specifically the mammalian glucose transporters Glut1 and Glut4, are presented with experimental data in which the accuracy and flexibility of the system are examined. This method reliably measures changes in the initial concentration for each of three target DNA sequences. The reaction is not acutely sensitive to variations in either the primer sites or internal sequence, and although the initial concentrations of the target DNAs did affect the relative amplification efficiencies, the effect was limited and did not prohibit quantification. This PCR system was able to reliably detect differences as little as 50% in the initial concentration of the Glut1 target DNA sequence. Therefore, with the appropriate controls, PCR can be extended to include the simultaneous quantification of more than one target DNA with a single internal control.