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RT-PCR Procedure (rt-pcr + procedure)
Selected AbstractsEffect of lactational exposure to 1,2,3,4- tetrachlorodibenzo-p-dioxin on cytochrome P-450 1A1 mRNA in the neonatal rat liver: Quantitative analysis by the competitive RT-PCR methodPEDIATRICS INTERNATIONAL, Issue 5 2001Yumi Kono AbstractBackground and Methods: The aim of this study was to assess the effect of lactational exposure to dioxins in neonates on the cytochrome P450 1A1 (CYP1A1) induction in the level of gene expression. Maternal rats were treated with a single dose of 50 or 100 ,mol/kg 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD), a low potent congener of dioxins, on the first day post-partum (day 1). Induction of CYP1A1 mRNA expression was quantitatively analyzed by the competitive reverse transcription,polymerase chain reaction (RT,PCR) method. Results: The CYP1A1 mRNA was detectable at extremely low amounts in the liver of control neonates and mothers. The mRNA ratios of CYP1A1 to ,-actin in neonates were dose-dependently increased by the treatment of 1,2,3,4-TCDD of their mothers. Its peak occurred on day 6 and was sustained at the same level on day 10. Increases of the ratio with 100 ,mol/kg 1,2,3,4-TCDD on day 2, 6 and 10 were 26-, 40- and 40-fold of the appropriate controls, respectively. These levels paralleled the activity of ethoxyresorufin-o-deethylase, representing CYP1A mediated monooxygenase. In the mother, the mRNA ratio was increased only to threefold of the control, 10 days after treatment. Conclusion: Current RT-PCR procedure enabled to assess both constitutive and induced levels of CYP1A1 mRNA in the neonatal rat livers. Although the dose of 1,2,3,4-TCDD selected in this study was about 5000 times higher than the daily intake of dioxins in breast-fed infants, CYP1A1 mRNA was highly induced for a longer period of time in neonatal rats receiving 1,2,3,4-TCDD via lactation than the treated maternal rats. [source] Serological and molecular detection of Tomato chlorosis virus and Tomato infectious chlorosis virus in tomatoPLANT PATHOLOGY, Issue 2 2009M. Jacquemond Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are two criniviruses inducing similar yellowing symptoms in tomato. An approximately 4 kb central region of the genomic RNA2 of French ToCV and TICV isolates was sequenced. TICV, for which no other sequences were available, appeared as a distant species in the genus, being close only to LIYV (Lettuce infectious yellows virus) for some, but not all, proteins. ToCV has more than 98% nucleotide identity with isolates from the US and Spain, and sequencing the CP gene of several isolates collected in different regions in southern France during 2 years suggested a unique origin. Polyclonal antisera were produced using capsid proteins of both viruses expressed in Escherichia coli. DAS-ELISA assays were developed for routine diagnosis and conditions for preparing samples for an optimized detection were determined. No cross-reactions were observed. However, some false-negative results, corresponding to samples giving ELISA readings close to the detection limit were regularly detected, particularly for ToCV (approximately 5% of the samples). A triplex RT-PCR assay was thus developed, which allowed detection of both viruses in a one-step protocol. An internal PCR control was included, which in addition showed that it could be used as a control for the entire RT-PCR procedure. Finally, combining DAS-ELISA in a first round, and triplex RT-PCR for doubtful samples, appeared the best way to achieve a reliable diagnosis of these viruses. [source] hTERT expression in sporadic renal cell carcinomasTHE JOURNAL OF PATHOLOGY, Issue 2 2001Valérie Paradis Abstract Human telomerase is a specialized reverse transcriptase that catalyses telomeric repeat addition at the ends of chromosomes. Activation of this enzyme is one of the key steps in cell immortalization and carcinogenesis, and one of its components, hTERT, is considered as the rate-limiting factor. While telomerase activity was found to be prognostically relevant in various cancers, results obtained from renal cell carcinomas (RCC) failed to show any correlation with the usual prognostic factors. The aim of the study was to reassess the role of telomerase and its hTERT component in the biological behaviour of RCC using new quantitative techniques, such as the quantitative evaluation of hTERT mRNA level by a real-time RT-PCR procedure and the mesuring of telomerase activity by an ELISA TRAP assay. Since experimental evidence supports a relationship between cell proliferation or c-myc expression and telomerase, the proliferation index and c-myc mRNA levels were also studied. Forty-one RCC (29 conventional renal cell carcinomas (CRCC), 10 papillary RCC and two urothelial carcinomas) were studied. In 73% of cases, normalized hTERT mRNA expression was significantly higher in the tumour sample than in the normal tissue. Telomerase activity was detected in 63% of RCC, while corresponding normal tissue was always negative. Analysis of correlations showed firstly that both telomerase activity and hTERT mRNA level were lower in the group of CRCC versus non-CRCC (TRAP: 0.3±0.1 versus 0.6±0.2, p<0.05; hTERT/PO mRNA: 5±3 versus 37±8, p<0.001, respectively); secondly, that in the group of CRCC, hTERT mRNA expression level was correlated with the stage of the tumour (p=0.01); and thirdly, that no correlation was observed between c-myc mRNA level and hTERT mRNA level. In conclusion, these results support the involvement of telomerase in RCC and the potential interest of hTERT mRNA quantification. Copyright © 2001 John Wiley & Sons, Ltd. [source] Technical limits of comparison of step-sectioning,immunohistochemistry and RT-PCR on breast cancer sentinel nodes: a study on methacarn-fixed tissueJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Lorenzo Daniele Abstract The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT-PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step-sectioning protocol at 100 micron-intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT-PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT-PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT-PCR negative cases, metastases were detected only on sections distant from those analysed by RT-PCR. CEA and/or CK19 were positive by RT-PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN. [source] | ||||||||||