Level Of DNA Contamination Research Articles

Non-Invasive Preimplantation Genetic Testing for Aneuploidy and the Mystery of Genetic Material: A Review Article.

This review focuses on recent findings in the preimplantation genetic testing (PGT) of embryos. Different preimplantation genetic tests are presented along with different genetic materials and their analysis. Original material concerning preimplantation genetic testing for aneuploidy (PGT-A) was sourced by searching the PubMed and ScienceDirect databases in October and November 2021. The searches comprised keywords such as ‘preimplantation’, ‘cfDNA’; ‘miRNA’, ‘PGT-A’, ‘niPGT-A’, ‘aneuploidy’, ‘mosaicism’, ‘blastocyst biopsy’, ‘blastocentesis’, ‘blastocoel fluid’, ‘NGS’, ‘FISH’, and ‘aCGH’. Non-invasive PGT-A (niPGT-A) is a novel approach to the genetic analysis of embryos. The premise is that the genetic material in the spent embryo culture media (SECM) corresponds to the genetic material in the embryo cells. The limitations of niPGT-A are a lower quantity and lesser quality of the cell-free genetic material, and its unknown origin. The concordance rate varies when compared to invasive PGT-A. Some authors have also hypothesized that mosaicism and aneuploid cells are preferentially excluded from the embryo during early development. Cell-free genetic material is readily available in the spent embryo culture media, which provides an easier, more economic, and safer extraction of genetic material for analysis. The sampling of the SECM and DNA extraction and amplification must be optimized. The origin of the cell-free media, the percentage of apoptotic events, and the levels of DNA contamination are currently unknown; these topics need to be further investigated.

Comparison of paperpoint and curette sampling of subgingival microbiome composition as analyzed by 16S rRNA gene amplicon sequencing

ABSTRACTObjective: Our aim was to compare the subgingival microbiome composition in samples collected by curettes and paperpoints.Methods: Subgingival plaque of rheumatoid arthritis patients with periodontitis (N=66) or gingivitis (N=15) was collected at two timepoints by sterile curettes and paperpoints. Unused sterile paperpoints were included as controls. The microbial DNA was processed for 16S rRNA gene amplicon sequencing. The data was processed and clustered into Operational Taxonomic Units (OTUs) and assessed by multivariate analyses.Results: Unused paperpoints showed various levels of DNA contamination. The OTUs specific for unused paperpoints were classified as Exiguobacterium, Enterococcus, Methylobacterium, Aquabacterium and Pseudomonas and were removed from the dataset.Microbial profiles of curette samples differed significantly from the paperpoint samples (PERMANOVA, p=0.0009, F=3.167). The paperpoint samples had significantly higher proportion of OTUs classified as Streptococcus, Gemella, Parvimonas, Haemophilus, Aggregatibacter and Clostridiales family XIII incertae sedis (p<0.05). Curette samples harbored higher proportion of Corynebacterium, Prevotella, Selenomonas, Actinomyces and Treponema (p<0.05).Curette samples had significantly higher species richness (p=0.01) and Shannon Diversity Index (p=0.009) than the paperpoint samples.Conclusions: Different subgingival plaque sampling techniques result in different microbiome profiles. Samples by paperpoints introduce microbial DNA contaminants and show underestimated microbial diversity compared to samples obtained by curettes.

COMPARISON OF RNA EXTRACTION METHODS FOR Passiflora edulis SIMS LEAVES

ABSTRACT Functional genomic analyses require intact RNA; however, Passiflora edulis leaves are rich in secondary metabolites that interfere with RNA extraction primarily by promoting oxidative processes and by precipitating with nucleic acids. This study aimed to analyse three RNA extraction methods, Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), TRIzol® Reagent (Invitrogen) and TRIzol® Reagent (Invitrogen)/ice -commercial products specifically designed to extract RNA, and to determine which method is the most effective for extracting RNA from the leaves of passion fruit plants. In contrast to the RNA extracted using the other 2 methods, the RNA extracted using TRIzol® Reagent (Invitrogen) did not have acceptable A260/A280 and A260/A230 ratios and did not have ideal concentrations. Agarose gel electrophoresis showed a strong DNA band for all of the Concert™ method extractions but not for the TRIzol® and TRIzol®/ice methods. The TRIzol® method resulted in smears during electrophoresis. Due to its low levels of DNA contamination, ideal A260/A280 and A260/A230 ratios and superior sample integrity, RNA from the TRIzol®/ice method was used for reverse transcription-polymerase chain reaction (RT-PCR), and the resulting amplicons were highly similar. We conclude that TRIzol®/ice is the preferred method for RNA extraction for P. edulis leaves.

Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays

Genome-wide expression studies of human blood samples in the context of epidemiologic surveillance are confronted by numerous challenges—one of the foremost being the capability to produce reliable detection of transcript levels. This led us to consider the Paxgene™ Blood RNA System, which consists of a stabilizing additive in an evacuated blood collection tube (PAX tube) and a sample processing kit (PAX kit). The PAX tube contains a solution that inhibits RNA degradation and gene induction as blood is drawn into the tube. The stability of RNA in PAX tubes under conditions for practical clinical applications has been determined by RT-PCR (Clin. Chem. 48 (2002) 1883), but has not been assessed at the transcriptome level on Affymetrix® microarrays. Here, we report a quality assured and controlled protocol that is capable of producing reliable gene expression profiles using the GeneChip® system with RNA isolated from PAX tubes. Using this protocol, we compared quality metrics and gene-expression profiles of RNA, extracted from blood in PAX tubes that sat at room temperature for 2 h, with that of blood in PAX tubes incubated at room temperature for 9 h followed by storage at −20 °C for 6 days. Of numerous metrics, differences between the two handling methods were detected for the level of DNA contamination, RNA yield, and double stranded cDNA yield. Analysis of variance of gene-expression revealed small but significant differences between the handling methods. These results contribute to the determination of protocols for clinical studies and progress us towards the goal of using the transcriptome in diagnosis and surveillance.

Interference of contaminating DNA in the quantification of a toluene-induced tod gene in Pseudomonas putida

To determine the extent of interference of co-extracted DNA contamination in the quantification of the tod gene transcript, two different concentrations of RNA (high, 500 ng/μl; low, 250 ng/μl) from a toluene-induced culture of Pseudomonas putida were treated with different amounts of DNase (2, 4, 6 and 8 U) and incubated for 30 and 60 min. The highly sensitive and reproducible TaqMan system was used to quantify the transcript of the tod gene, the tod gene in contaminating DNA and the 16S rRNA gene in DNase-treated RNA samples. For the high RNA concentration, the shorter incubation time (30 min) lowered the level of contaminating DNA as evidenced by the presence of 2.5 × 10 6 copies of the tod gene before treatment to 1.4 × 10 5 copies/μl (8 U), whereas, irrespective of the DNase units used, the longer incubation time (60 min) considerably lowered the level of DNA contamination (2.5 × 10 6 to 6.5 × 10 2 copies of the tod gene/μl). However, for the low RNA concentration, DNase treatment was found to be equally effective in lowering the level of contaminating DNA (10 6 to 10 2 copies of the tod gene/μ), irrespective of the incubation time and the amount of DNase used. Although the results of gel electrophoresis of conventional PCR amplification of the low RNA concentration revealed the absence of the target gene in contaminating DNA, the results of the TaqMan PCR indicated that a very low amount of contaminating DNA (less than 10 3 copies of the tod gene/μl) was still present in RNA samples, even after the DNase treatment. The number of copies of the tod gene transcript in RNA samples did not show any marked variation because of the DNase treatment. However, the proportion of contaminating DNA in RNA samples considerably decreased due to the treatment (0.01 to 0.000001). Furthermore, these results suggested that the extent of the removal of contaminating DNA from RNA samples depends on the concentration of RNA, the amount of DNase used and the incubation time. It is also suggested that the copies of the catabolic genes in contaminating DNA have to be quantified along with the target genes in RNA samples to have a more accurate quantification of the target genes for better understanding of their roles in many microbial processes.

An Assessment of DNA Contamination Risks in New York City Medical Examiner Facilities

DNA evidence holds an important position in criminal investigations and proceedings. The polymerase chain reaction (PCR) is often utilized to amplify polymorphic regions of DNA which are subsequently typed to produce distinct genotypes. The sensitivity of PCR-based techniques provides a major advantage over other DNA or conventional serological typing systems. Samples containing quantities of DNA in the picogram range are often typed. However, the unprecedented sensitivity of PCR is often cited as a criticism. One concern is that the interpretation of PCR typing can be affected by DNA contaminants from foreign sources. In this report, the level of DNA contamination in New York City Medical Examiner facilities and its potential affects on HLA-DQA1 typing were assessed. Two related studies conducted over a five week period measured and typed HLA-DQA1 from accumulated DNA on autopsy room and Forensic DNA Laboratory structures. The potential for DNA contamination from airborne sources was also evaluated in the autopsy suites. This study demonstrated the presence of small amounts of DNA on structural surfaces, but little evidence of airborne DNA contamination.

The estimation of DNA contamination in RNA and enzyme preparations

Commercially available RNA and enzyme preparations are monitored for DNA contamination. The method used involves reaction with p-nitrophenylhydrazine and separation of the reaction products by thin-layer chromatography. When this technique is used, RNA preparations and bovine pancreas ribonuclease are shown to have low levels of DNA contamination. An independent method, based on the enhanced fluorescence of DAPI in the presence of DNA, also detected DNA in the RNA preparations but not in the ribonuclease.