Article

Irreversible heat inactivation of DNAse I without RNA degradation

Catholic University of Leuven, Heverlee, Belgium.
BioTechniques (Impact Factor: 2.95). 09/2000; 29(2):252-4, 256.
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    • "The total RNA was incubated with 10 U/ml of DNase I at 37 C for 20 min in 20 mL of final reaction. At the end, the reaction was heated 5 min at 75 C (Wiame et al., 2000) and stored at À20 C until use. The extracted RNA was then quantified using a micro-volume spectrophotometer (NanoDrop) with 2 mL of sample and the relation 260/280 was more than 1.8. "
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    ABSTRACT: Organophosphate insecticides (OI) are widely used. To humans the main routes of exposure are skin and inhalation. For this, keratinocytes (HaCaT) and bronchial cells (NL-20) were used as cell culture models to evaluate the effects of OI. The aim of this study was to evaluate the effect of four OI on HaCaT and NL-20 cells: azinphos-methyl, (AM); parathion-methyl (PM); omethoate (OM); and methamidophos (MET). Cells were exposed to 0.1, 1 and 10 μg/μL of each. Results showed a decrease in cell viability in both cell lines. Viability of the NL-20 cell line decreased with the three concentrations of OM. All differences were significant (p < 0.05). Genotoxic damage, evaluated through the comet assay, was observed in both cell lines with AM. NL-20 cell line was more sensitive than HaCaT. Higher concentrations of the insecticides except MET, induced cell death. MET caused DNA damage in HaCaT cells at all concentrations. Differences were significant (p < 0.05). Both cell lines revealed the presence of single membrane vacuoles of different sizes when exposed to 1 μg/μL of each insecticide. Quantitative real time-polymerase chain reaction (RT-qPCR) showed an increase of BN1 gene in HaCaT by effect of AM and MET at 1 μg/μL. In conclusion, all the insecticides induced different levels of cyto and genotoxic effects in both cell lines.
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    • "Total RNA was isolated from the liver tissues of homogenized O. niloticus using The GeneJET TM RNA Purification Kit (Thermo Scientific #K0731) and according to theChomczynski andSacchi (1987) andBoom et al., (1990)method. Total cDNA for real time PCR reactions were generated from 1 lg DNase treated total RNA from all samples using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific #K1621) as described by the manufacturer according toWiame et al. (2000). "
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    ABSTRACT: The present study was undertaken to evaluate the therapeutic efficacy of reduced glutathione (GSH) and its curative prospective for the alleviation of aflatoxicosis in Oreochromis niloticus. 180 apparently healthy O. niloticus were assigned to six groups (A-F); A and B were kept as a negative and positive control of AFB1. Fish in groups C and D were injected with GSH at 3 and 6mg/kg body weight, BW, respectively), while those in groups E and F were injected at the start of the experiment by AFB1 (6mg/kg BW) then at the 7th day, they were injected with GSH at two doses. The results showed that AFB1 has a significant potency for increasing plasma creatinine and uric acid and for reducing the values of plasma urea and hepatic antioxidants. In addition, AFB1 reflected an up-regulation in the expression of the hepatic gene of glutathione peroxidase (GPx) and cytochrome CYP1A. The therapeutic efficacy of GSH against aflatoxicosis was recorded at the low level of GSH that improved values of both uric acid and hepatic antioxidants. In addition to that, GSH reflected up-regulated and down-regulated GPx and CYP1A gene expressions, respectively in fish group E, which gave a splendid indicator of the positive ability of GSH in the detoxification of aflatoxin in O. niloticus, while the optimal level of GSH antioxidant for detoxification of aflatoxin effects may need more studies.
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    • "Total RNA was isolated using the RNEASY® Plus Mini Kit (Qiagen, Hilden, Germany) from 60–80 mg of leaf, pseudostem, corm, and root tissue of in vitro regenerated plantlets maintained at room temperature. One microgram of RNA was used for cDNA synthesis with the REVERTAID™ H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) using an oligo(dT)18 primer or the luc+ sequence-specific Luc+R primer to detect transcription of the housekeeping actin (act) gene [52] or the luc+ transgene, respectively. The RT-PCR reaction followed the cycling program: initial denaturation at 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, and 68°C for 30 s with a final elongation step of 68°C for 2 min. "
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    ABSTRACT: Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc+) gene and low temperature-responsive luciferase activation was monitored in real time. Around 16,000 transgenic cell colonies were screened for baseline luciferase activity at room temperature 2 months after transformation. After discarding positive colonies, cultures were re-screened in real-time at 26 degrees C followed by a gradual decrease to 8 degrees C. The baseline activation frequency was 0.98%, while the frequency of low temperature-responsive luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies with luciferase activity responsive to low temperature were regenerated to plantlets and luciferase expression patterns monitored during different regeneration stages. Twenty four banana DNA sequences flanking the right T-DNA borders in seven independent lines were cloned via PCR walking. RT-PCR analysis in one line containing five inserts allowed the identification of the sequence that had activated luciferase expression under low temperature stress in a developmentally regulated manner. This activating sequence was fused to the uidA reporter gene and back-transformed into a commercial dessert banana cultivar, in which its original expression pattern was confirmed. This promoter tagging and real-time screening platform proved valuable for the identification of novel promoters and genes in banana and for monitoring expression patterns throughout in vitro development and low temperature treatment. Combination of PCR walking techniques was efficient for the isolation of candidate promoters even in a multicopy T-DNA line. Qualitative and quantitative GUS expression analyses of one tagged promoter in a commercial cultivar demonstrated a reproducible promoter activity pattern during in vitro culture. Thus, this promoter could be used during in vitro selection and generation of commercial transgenic plants.
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