Article

Irreversible heat inactivation of DNAse I without RNA degradation

Catholic University of Leuven, Heverlee, Belgium.
BioTechniques (Impact Factor: 2.75). 09/2000; 29(2):252-4, 256.
Source: PubMed
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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.
    BMC Plant Biology 07/2009; 9:77. DOI:10.1186/1471-2229-9-77 · 3.94 Impact Factor
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    • "We have consistently found a decrease in amplified cDNA in DNase-treated samples, particularly when performing RNA isolation with traditional methods (unpublished results), even when a chelating agent was added prior to the heating step. Incomplete RNA recovery after DNase inactivation in the presence of EDTA was not evident in past reports, due to the use of non-quantitative methods of nucleic acids analysis [35]. Our real-time PCR results, however, agree with numerous more recent findings [see ref. [36] for an overview of DNase-related problems]. "
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    ABSTRACT: Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost. This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres. This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.
    BMC Biotechnology 02/2005; 5(1):2. DOI:10.1186/1472-6750-5-2 · 2.59 Impact Factor
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    • "In these organisms, the only efficient method to eliminate genomic or plasmid DNA is by exhaustively treating the RNA template with DNase. However, DNase treatment and the subsequent inactivation of the enzyme may cause RNA loss due to the presence of divalent cations in the buffer and/or due to phenol/chloroform extraction (Wiame et al., 2000). This is particularly troublesome when small amounts of RNA, such as those derived from multi-gene polycistronic operons are analyzed. "
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    ABSTRACT: RT-PCR is a powerful technique used in the amplification and detection of rare mRNAs. However, one of the most serious drawbacks of this method is the amplification of false-positive products due to DNA contamination in the RNA samples. This pitfall is particularly hard to overcome when RNA from prokaryotic origin is used. We present here a modification of the EXACT RT-PCR method that was successfully employed in the amplification of the low abundant full-length polycistronic pst operon mRNA of Escherichia coli. No DNase treatment of the RNA template is required, but unlike the original EXACT RT-PCR, a hybrid primer that is not composed of oligo(dT) was used. A nonhomologous sequence was incorporated at the reverse transcription step into the 5' end of the first-strand cDNA by means of the hybrid primer. For the PCR, a gene-specific primer and a second primer identical to the nonhomologous portion of the hybrid primer were used. To avoid amplification of genomic DNA, the hybrid-primer molecules that were not incorporated into the first-strand cDNA were removed by RNase H treatment followed by ultrafiltration.
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