Mechanisms of degradation of DNA standards for calibration function during storage

Rossmanith, Peter; Röder, Barbara; Frühwirth, Karin; Vogl, Claus; Wagner, Martin

doi:10.1007/s00253-010-2943-2

Cited by 28 publications

(37 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, different samples failed the two PCR and pyrosequencing assays suggesting that the source of gDNA played an important role in the success of the analysis and the storage length did not seem to have a major impact. This is in line with previous observations [37,41] that gDNA fragmentation over time with storage has little impact on short DNA detection (200-700 bp). The variation observed in the PCR success rate might be dependent on the amount of natural PCR inhibitors (protein, haemoglobin, iron) present in the newborn DBS [40].…”

Section: Discussionsupporting

confidence: 93%

Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue

et al. 2013

View full text Add to dashboard Cite

BackgroundGenotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis.All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort.ResultsBoth whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples.ConclusionsgDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies.

show abstract

Section: Discussionsupporting

confidence: 93%

Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue

et al. 2013

View full text Add to dashboard Cite

show abstract

“…If not otherwise stated, 10 µl of the purified DNA were used as template in the real-time PCR. Due to shearing and depurination of DNA, the quality of the DNA standard decreases with time, even when stored frozen [10]. Thus, the DNA standard (e.g.…”

Section: Methodsmentioning

confidence: 99%

“Limits of Control” – Crucial Parameters for a Reliable Quantification of Viable Campylobacter by Real-Time PCR

Krüger

Buhler

Iwobi³

et al. 2014

PLoS ONE

View full text Add to dashboard Cite

The unsuitability of the “CFU” parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.

show abstract

“…In these cases, the use of RNAlater or ethanol is more feasible and reliable. Additionally, even if freezers are available, not all commercial shippers allow the use of dry ice so the risk of samples thawing or going through freeze-thaw cycles during transport must be considered [277].…”

Section: Resultsmentioning

confidence: 99%

Co-evolution in context: The importance of studying gut microbiomes in wild animals

Amato

2013

Microbiome Science and Medicine

150

152

View full text Add to dashboard Cite

IntroductionAs sequencing technology makes data generation faster, cheaper, and more comprehensive, studies of gut microbial communities are multiplying at an astonishing rate. As a result, our understanding of the host-gut microbe relationship is constantly improving. Studies to date have demonstrated that the gut microbiota contributes to host nutrition, health and behavioral patterns by providing energy and nutrients, improving immune function, and influencing the production of neuroactive molecules [1][2][3][4][5][6][7][8][9][10][11][12]. Changes in the composition of the gut microbial community are known to lead to changes in its function, which can alter host nutrition, health and behavior [6,[13][14][15][16][17][18][19][20][21][22][23]. Environmental factors such as diet or social contact are largely responsible for determining the composition of the gut microbial community [24][25][26][27][28][29][30][31], but host genotype also affects the abundances of some microbial genera [28,32,33].Because host-gut microbe relationships are influenced to some extent by host genotype, and gut microbial community composition differs according to host phylogeny [34][35][36], discussions of the co-evolution of host and gut microbiota are common in the current literature [7,[34][35][36][37]. Some researchers argue that since microbes are found in animals as simple as earthworms, the co-evolution of animals and bacteria has been Co-evolution in context: The importance of studying gut microbiomes in wild animals Abstract Because the gut microbiota contributes to host nutrition, health and behavior, and gut microbial community composition differs according to host phylogeny, co-evolution is believed to have been an important mechanism in the formation of the host-gut microbe relationship. However, current research is not ideal for examining this theme. Most studies of the gut microbiota are performed in controlled settings, but gut microbial community composition is strongly influenced by environmental factors.To truly explore the co-evolution of host and microbe, it is necessary to have data describing host-microbe dynamics in natural environments with variation in factors such as climate, food availability, disease prevalence, and host behavior. In this review, I use current knowledge of host-gut microbe dynamics to explore the potential interactions between host and microbe in natural habitats. These interactions include the influence of host habitat on gut microbial community composition as well as the impacts of the gut microbiota on host fitness in a given habitat. Based on what we currently know, the potential connections between host habitat, the gut microbiota, and host fitness are great. Studies of wild animals will be an essential next step to test these connections and to advance our understanding of host-gut microbe co-evolution. KeywordsGut microbiota • host-microbe • co-evolution • habitat • ecology • fitness occurring for more than 800 million years [38,39]. Additionally, the increased complexity and stabilit...

show abstract

Mechanisms of degradation of DNA standards for calibration function during storage

Cited by 28 publications

References 37 publications

Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue

Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue

“Limits of Control” – Crucial Parameters for a Reliable Quantification of Viable Campylobacter by Real-Time PCR

Co-evolution in context: The importance of studying gut microbiomes in wild animals

Contact Info

Product

Resources

About