Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

Hashemi-Petroudi, Seyyed Hamidreza; Nematzadeh, Ghorbanali; Ahmadian, Gholamreza; Yamchi, Ahad; Kuhlmann, Markus

doi:10.3791/55451

Cited by 14 publications

(20 citation statements)

References 13 publications

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“…Although trends in total RNA degradation tend to follow those found using mRNA transcripts, the latter is more sensitive as it targets a specific gene and can determine actual copy numbers [3,17,21]. It is also susceptible to genomic DNA contamination [40]. Fu et al.…”

Section: Discussionmentioning

confidence: 99%

Using total RNA quality metrics for time since deposition estimates in degrading bloodstains

Elliott

Stotesbury

Shafer

2021

Preprint

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Determining the age, or time since deposition (TSD), of bloodstains would provide forensic scientists with critical information regarding the timeline of the events of bloodshed during a crime. The physicochemical changes that occur to major biomolecules as a bloodstain dries can be used to estimate the TSD of bloodstains. For example, high-resolution automated gel electrophoresis can be used to quantify the timewise degradation of DNA present in bloodstains. Our study aims to analyze and quantify the timewise degradation trends found in total RNA from bloodstains, expanding the scope of the TSD research which has previously explored DNA and targeted mRNA molecules. Fifty bloodstains were stored in plastic microcentrifuge tubes at room temperature and tested over 10 different timepoints spanning one week. A total of eight RNA metrics were visually assessed and quantified using linear regression. RNA Integrity Number equivalent (RINe), total RNA concentration, and 28S/18S rRNA peak area ratios were retained for further analyses based on their relationship with time and limited correlations. RINe and total RNA concentration both exhibited negative trends over time, highlighting a decrease in quality and quantity. RINe was the RNA metric that demonstrated the greatest association with time (R2 = 0.696). Generalized linear mixed-effects models including donor (biological replicate) as a random effect increased the fit for all RNA metrics to varying degrees, but no significant differences were found between biological replicates for the RINe metric. Our results illustrated the presence of a significant decrease in the retained RNA metrics after 24 hours, suggesting that this method could be used to reliably differentiate day-old bloodstains from older bloodstains. Future work should focus on recreating this study in different environmental conditions, including testing on a variety of substrates.

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Section: Discussionmentioning

confidence: 99%

Using total RNA quality metrics for time since deposition estimates in degrading bloodstains

Elliott

Stotesbury

Shafer

2021

Preprint

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“…Other methods such as mRNA detection by RT-(q)PCR or functional assessment using agonistic or antagonistic ligands also bear uncertainties. RT-PCR is a very sensitive detection method, but, besides the risk that trace amounts of contaminating genomic DNA may pose the presence of specific mRNA [ 117 ], it is not known which degree of mRNA expression would enable effective receptor expression [ 118 ]. The use of receptor ligands is afflicted with problems similar to those of the antibodies; selectivity, which is strictly concentration-dependent, is the major concern [ 18 , 119 ].…”

Section: Histamine Receptors In the Intestinementioning

confidence: 99%

The Function of the Histamine H4 Receptor in Inflammatory and Inflammation-Associated Diseases of the Gut

Schirmer

Neumann

2021

IJMS

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Histamine is a pleiotropic mediator involved in a broad spectrum of (patho)-physiological processes, one of which is the regulation of inflammation. Compounds acting on three out of the four known histamine receptors are approved for clinical use. These approved compounds comprise histamine H1-receptor (H1R) antagonists, which are used to control allergic inflammation, antagonists at H2R, which therapeutically decrease gastric acid release, and an antagonist at H3R, which is indicated to treat narcolepsy. Ligands at H4R are still being tested pre-clinically and in clinical trials of inflammatory diseases, including rheumatoid arthritis, asthma, dermatitis, and psoriasis. These trials, however, documented only moderate beneficial effects of H4R ligands so far. Nevertheless, pre-clinically, H4R still is subject of ongoing research, analyzing various inflammatory, allergic, and autoimmune diseases. During inflammatory reactions in gut tissues, histamine concentrations rise in affected areas, indicating its possible biological effect. Indeed, in histamine-deficient mice experimentally induced inflammation of the gut is reduced in comparison to that in histamine-competent mice. However, antagonists at H1R, H2R, and H3R do not provide an effect on inflammation, supporting the idea that H4R is responsible for the histamine effects. In the present review, we discuss the involvement of histamine and H4R in inflammatory and inflammation-associated diseases of the gut.

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“…Contaminant DNA might be co-amplified together with the cDNA, leading to background and false positive results, which is, for example, a problem in metagenomics analyses [1]. To determine the amount of contaminating genomic DNA in RNA samples, several PCR-based approaches have been proposed, e.g., the addition of a genomic DNA reference sample [2] and the detection of the intron/exon ratio of a housekeeping gene [3] and of ribosomal DNA [4].…”

Section: Introductionmentioning

confidence: 99%

Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR

Ahlemeyer

Colasante

Baumgart-Vogt

2020

MPs

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In transfection experiments with mammalian cells aiming to overexpress a specific protein, it is often necessary to correctly quantify the level of the recombinant and the corresponding endogenous mRNA. In our case, mouse calvarial osteoblasts were transfected with a vector containing the complete Pex11β cDNA (plasmid DNA). The Pex11β mRNA level, as calculated using the RT-qPCR product, was unrealistically higher (>1000-fold) in transfected compared to non-transfected cells, and we assumed that there were large amounts of contaminating plasmid DNA in the RNA sample. Thus, we searched for a simple way to distinguish between plasmid-derived mRNA, endogenous genome-derived mRNA and plasmid DNA, with minimal changes to standard RT-PCR techniques. We succeeded by performing a plasmid mRNA-specific reverse transcription, and the plasmid cDNA was additionally tagged with a nonsense tail. A subsequent standard qPCR was conducted using appropriate PCR primers annealing to the plasmid cDNA and to the nonsense tail. Using this method, we were able to determine the specific amount of mRNA derived from the transfected plasmid DNA in comparison to the endogenous genome-derived mRNA, and thus the transfection and transcription efficiency.

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Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

Cited by 14 publications

References 13 publications

Using total RNA quality metrics for time since deposition estimates in degrading bloodstains

Using total RNA quality metrics for time since deposition estimates in degrading bloodstains

The Function of the Histamine H4 Receptor in Inflammatory and Inflammation-Associated Diseases of the Gut

Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR

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