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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    ABSTRACT: Chloroplast RNA splicing is usually studied by complicated methods, such as Northern blot hybridization, RNAse protection, or primer extension assays. The use of a simpler RT-PCR technique frequently results in the underestimation of unspliced pre-mRNA levels. Five protein-coding genes from the maize plastome were studied to analyze which factors can lead to an apparent reduction in the pool of intron-containing transcripts compared to mature RNA. It was found that the accurate determination of the unspliced RNA level should take into account the DNase inactivation mode, the cDNA synthesis temperature, and the type of DNA polymerase. For one gene, the portion of unspliced RNAs decreased with increasing number of PCR cycles. It was found that intron-containing and intron-free strands of amplicons can form heteroduplexes after PCR. A simple and effective technique of investigating chloroplast spliced mRNA and unspliced pre-mRNA is suggested based on the obtained data.
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    ABSTRACT: In theory, RNA expression patterns, including the presence and relative abundance of particular RNA species, provide cell and tissue specific information that could be of use to forensic scientists. An mRNA based approach could allow the facile identification of the tissue components present in a body fluid stain and conceivably could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory. Some of the potential advantages include greater test specificity, and the ability to perform simultaneous analysis using a common assay format for the presence of all body fluids of forensic interest. In this report, the recovery and stability of RNA in forensic samples was evaluated by conducting an in-depth study on the persistence of RNA in biological stains. Stains were prepared from blood, saliva, semen, and vaginal secretions, and were exposed to a range of environmental conditions so that the affects of different light sources, temperatures, and environments could be assessed. Using the results from quantitation and sensitivity studies performed with pristine forensic stains, the RNA stability of samples which were collected over a period of 1 day to 1 year for blood, saliva, and vaginal secretion stains and for up to 6 months for semen stains were analyzed. The extent of RNA degradation within each type of body fluid stain was determined using quantitation of total RNA and reverse transcriptase polymerase chain reaction (RT-PCR) with selected housekeeping and tissue-specific genes. The results show that RNA can be recovered from biological stains in sufficient quantity and quality for mRNA analysis. The results also show that mRNA is detectable in samples stored at room temperature for at least one year, but that heat and humidity appear to be very detrimental to the stability of RNA.