TaqMan RT-PCR Assay Research Articles

Development of a semi-quantitative real-time RT-PCR for the detection of measles virus

Real-time detection of polymerase chain reactions allows convenient detection and quantification of virus-derived nucleic acids in clinical specimens. We have developed a real-time RT-PCR assay for the detection of measles virus (MV) genomic RNA, and compared it to a well-established conventional RT-PCR assay. Based on a serial dilution of the live-attenuated MV Edmonston Zagreb vaccine, the detection limits were approximately 0.1 and 0.02 cell culture infectious dose 50% units (CCID50) per test for the conventional and TaqMan RT-PCR assays, respectively. Furthermore, tissue materials spiked with known quantities of MV were equally well detected in both assays. The TaqMan assay was linear within a range of 10(4.4) to 10(-0.6)CCID50/ml, with an intra-assay variability lower than 3% and an inter-assay variability ranging from 1.5% at 10(4.4)CCID50/ml to 8.7% at 10(-0.6)CCID50/ml. The TaqMan assay could detect representative wild-type viruses from the currently active MV clades, and could detect MV genome in clinical specimens obtained from measles patients. Finally, quantification of MV RNA in peripheral blood mononuclear cells or broncho-alveolar lavage cells from cynomolgus macaques collected at different time points after experimental infection showed a good correlation with virus isolation data. In conclusion, the TaqMan assay developed is specific, sensitive, rapid and reproducible, and can be of use for diagnostic purposes or for studies on the pathogenesis of measles.

TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type

TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type

Differential effects of acute and chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarray.

Studies were performed to determine the effects of acute and chronic voluntary periods of exercise on the expression of hippocampal genes. RNAs from rodents exposed to a running wheel for 3, 7 and 28 days were examined using a microarray with 1176 cDNAs expressed primarily in the brain. The expression of selected genes was quantified by Taqman RT-PCR or RNase protection assay. The largest up-regulation was observed in genes involved with synaptic trafficking (synapsin I, synaptotagmin and syntaxin); signal transduction pathways (Ca2+/calmodulin-dependent protein kinase II, CaM-KII; mitogen-activated/extracellular signal-regulated protein kinase, MAP-K/ERK I and II; protein kinase C, PKC-delta) or transcription regulators (cyclic AMP response element binding protein, CREB). Genes associated with the glutamatergic system were up-regulated (N-methyl-d-aspartate receptor, NMDAR-2A and NMDAR-2B and excitatory amino acid carrier 1, EAAC1), while genes related to the gamma-aminobutyric acid (GABA) system were down-regulated (GABAA receptor, glutamate decarboxylase GAD65). Brain-derived neurotrophic factor (BDNF) was the only trophic factor whose gene was consistently up-regulated at all timepoints. These results, together with the fact that most of the genes up-regulated have a recognized interaction with BDNF, suggest a central role for BDNF on the effects of exercise on brain plasticity. The temporal profile of gene expression seems to delineate a mechanism by which specific molecular pathways are activated after exercise performance. For example, the CaM-K signal system seems to be active during acute and chronic periods of exercise, while the MAP-K/ERK system seems more important during long-term exercise.

Improved detection of Sugarcaneyellowleafvirus using a real-time fluorescent (TaqMan) RT-PCR assay

Yellow leaf syndrome (YLS) of sugarcane has been associated with Sugarcane yellow leaf virus (ScYLV) and has been reported from most sugarcane growing countries around the world. As sugarcane is vegetatively propagated, it is important to use effective and sensitive detection methods to screen new propagating material. Virus detection in symptomatic tissue is currently achieved using enzyme linked immunosorbent assay (ELISA), tissue blot immunoassay (TBIA) or a conventional RT-PCR based assay. This paper reports the development of an improved assay based on multiplex real-time fluorescent RT-PCR. The new assay is 100-fold more sensitive than conventional RT-PCR, and incorporates a novel ‘RNA specific’ internal positive control (based around the intron of the caffeic acid 3- o-methyltransferase gene) to guard against false negative results. The paper also describes the comparison of eight RNA extraction methods for sugarcane tissue giving a number of alternatives for different laboratory situations. The sensitivity of this assay has allowed the detection of ScYLV in many samples that were thought to be healthy following conventional testing (RT-PCR, ELISA or TBIA). The detection of ScYLV using this TaqMan assay can be applied to the production of ScYLV-free plants and prevents its spread through the propagation material.

Up-regulation of vitamin D3 up-regulated protein 1 gene in response to 5-fluorouracil in colon carcinoma SW620

Despite the wide use of 5-fluorouracil (5-FU) for colon cancer, the genes regulating its cytotoxic effect are poorly understood. We used a high-density oligonucleotide microarray representing approximately 7000 genes to determine changes in gene expression caused by 5-FU treatment in the colon cancer cell line, SW620. The microarray showed that the most strongly up-regulated gene by 5-FU was vitamin D3 up-regulated protein 1 (VDUP1), an interesting stress response gene, which was originally reported as a vitamin D3 inducible gene in HL-60. TaqMan RT-PCR assay confirmed that VDUP1 gene expression was significantly increased after 24 h of 5-FU treatment compared with untreated control (p<0.01). Moreover, the expression of vitamin D3 receptor, thymidylate synthase (TS), and E2F1 did not change within 24 h of 5-FU treatment, suggesting a different gene-regulatory pathway from that of VDUP1. Recent studies have gradually clarified the potential role of VDUP1 via interaction with TRX in an anti-tumor effect. Therefore, VDUP1 not only may be induced by stress response as a result of 5-FU cytotoxicity, but may also play a key role in 5-FU cytotoxicity in colon cancers. Our experiment using a microarray and TaqMan RT-PCR assay, together with previous reports, provides new insight into a potential mechanism of 5-FU cytotoxicity.

Vascular endothelial growth factor-C (VEGF-C) overexpression is correlated with poor prognosis in intrahepatic cholangiocarcinoma (CCC)

Background: METI-I-1 and METI-I-2 are newly identified gene that show sequence similarity to metalloproteinase, disintegrin and TSP type1 repeats. METH-1 and -2 have antiangiogenic properties and murine homotogue of METH-1 shows metalloproteinese function. However, the significance of METH-1 and -2 has not yet been determined in human solid tumors. In this study, we examined METH-1 and -2 mRNA expressions in two tumors with distinct differences in vasculadty, pancreatic ductal adenocarcinoma and hepatocelluier carcinoma (HCC). Methods: METH-1 and -2 mRNA expressions were identified in 6 pancreatic cancer cell lines, and then examined in surgically resected samples of 32 pancreatic cancer, 20 pancreas, 16 HCC and 16 liver cirrhosis. METH-1 and -2 gene expressions were measured by RT-PCR and quantltated by TaqMan RT-PCR assay using beta-actin as an internal standard and expressed as a METH-1/beta-actin or METH-2/beta-actin mRNA ratio. Vascularity of tumor was estimated by CD34 staining. Correlation of METH-1 mRNA expression with clinicopathological factors were further analyzed in 32 patients with pancreatic cancer. Results: 4 out of 6 pancreatic cancer cell lines expressed METH-1 and none expressed METH-2. METH-1 mRNA was substantially expressed in both pancreatic cancer and pancreas tissue, hot METH-2 did not. When quantitated with TaqMan RT-PCR, METH-1 mHNA expression in pancreatic cancer tissue was significantly lower than that in non-cancerous lesion of pancreas (p = 0.002), and similar results were obtained between HCC and cirrhotic liver (p = 0.003). Reduction of METH1 mRNA expression in pancreatic cancer was nearly equal with that in HCC. The expression of METH-1 had no significant correlation with MVD (Mean Vascular Density) in pancreatic cancer tissue and HOG. However, pancreatic cancer with high expression of METH-1 significantly increased lymph node metastasis or severe retroperitoneal invasion (p=0.033 and p = 0.039, respectively). There were no significant correlations between METH-1 expression and tumor size or histological grade. Patients with high METH-1 expression showed significantly worse prognosis than those with low METH-1 (p=0.032). Conclusions: METH-f, which is initially reported to have a very potent antiangiogenetic property, does not seem to be a predominant factor to determine the tumor vascularity in pancreatic cancer and HCC, but lowered expression of METH-1 might be involved in tumor angiogenesis. METH-1 is rather involved in progression of pancreatic cancer through local invasion and lymphnode metastasis.

Development of a novel real-time RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues

Pigs are more difficult to immunise and more variable in their response to foot-and-mouth disease (FMD) than other livestock species. This has important consequences for FMD control during both prophylactic vaccination programmes in endemic situations and when emergency vaccination may be used as an adjunct to stamping out during outbreaks in countries normally free from the disease. The rapid and effective control of FMD in pigs is especially important in regions of high pig density since infected pigs have the potential to generate plumes of airborne virus and spread infection beyond the immediate control area. Increased knowledge of the kinetics of FMDV replication in pigs, especially in their respiratory tracts, could create opportunities for strategies to improve FMD vaccines for pigs. A fluorogenic TaqMan RT-PCR assay specific for the IRES (internal ribosome entry site) sequence of the O 1 Kaufbeuren/Lausanne strain of FMDV has been developed for this purpose. The assay had a sensitivity of 0.1 TCID 50/ml, and a dynamic range of at least eight orders of magnitude. It was found that an established method of quantitation, relative to a housekeeping gene, exhibited two significant shortcomings when applied to a set of 16 anatomically highly diverse solid tissues: (i) tissue differences in housekeeping gene expression caused errors of up to 60-fold in estimated FMDV concentrations; and (ii) variability in total RNA yields caused unpredictable saturation of RT reactions, which in turn caused errors of up to 250-fold in the estimated FMDV concentration. A novel quantitation strategy, designated the C t min method, was developed to overcome these problems. The C t min method was based on the the RT-PCR examination of a dilution series of spectrophotometrically quantitated total RNA, spanning the optimum for the RT-PCR system used. The new method was influenced minimally by any tissue-specific RT-PCR inhibitors and was used to determine FMDV concentrations in tissues from four experimentally infected pigs. The results suggest that the lungs play a less important role in the replication of FMDV in pigs than was thought previously.

Quantitative reverse transcription-PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system

We established the validity of a quantitative reverse transcription (RT)-PCR assay for the RNA component of human telomerase (hTR), using the TaqMan fluorogenic detection system. Using this assay, we quantified hTR expression in two human pancreatic cancer cell lines, ASPC-1 and MIAPaCa-2. Our results indicated that hTR expression in MIAPaCa-2 was 1.99-fold higher than that in ASPC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the amount of hTR in clinical specimens.