DNA Extraction Procedure Research Articles

A systematic evaluation of ‘Bidi – a hand-rolled cigarette’ as a forensic DNA evidence

Bidis are small handmade cigarettes consisting of ~0.2 g of tobacco flakes rolled in a dried leaf of ‘Tendu' (Diospyros melanoxylon) or Piliostigma racemosum and tied with a thin thread. They have gained worldwide popularity among smokers and are often collected as forensic DNA evidence from crime scenes and processed similarly to cigarette butts. However, bidi’s composition and manufacturing process differs distinctly from the cigarette, and hence the optimal processing for DNA analysis should not be assumed to be similar to cigarette butts. In the present study, the methodical evaluation of the bidi for DNA analysis is reported and an additional process of cell elution from bidi stubs prior to DNA extraction is systematically compared with the direct lysis of bidi stubs which is identical to the standard practice in forensic labs for cigarette butts. In terms of cell recovery from bidi stubs, the SDS based Cell Elution Buffer (CEB) proved to be better than Tween20 based CEB. The three components (cell-elute, supernatant, and processed stub) obtained after the cell elution process of smoked bidi stubs showed varying amounts of DNA. The cell-elute and processed stub exhibited high quality DNA, resulting in 90–100% of the samples giving a full STR profile. On the contrary, the directly lysed stubs yielded high quantity but low quality of DNA, resulting in only 40% of the samples with full STR profiles. The cell elution process enables three components namely cell-elute, supernatant and processed stub from the same bidi to be used for forensic DNA analysis, although the cell-elute proved to be the best source of DNA for STR profiling. The current study demonstrates that the additional cell elution process proved to be an essential step prior to DNA extraction procedure for bidi stubs.

Comparison of methods for isolating fungal DNA

ObjectivesThe main aim of this work was to compare the methods of DNA isolation in the moulds of genus Mucorales with special regard to the amount and purity of the DNA acquired. The acquired DNA was then amplified by specific real-time PCR. DesignFive DNA extraction procedures were carried out in a Class 2 Biosafety cabinet in a dedicated room with suitable biosafety precautions and appropriate biowaste disposal methods. A total of 6 Mucorales clinical strains were used. ResultsFrom the viewpoint of concentration and purity, methods A shown abundant amount of fungal DNA whereas methods E report a pure fungal DNA with R260/280 of 1.7 near the optimal 1.8. The DNA quantity reach statistically difference at ANOVA test with p value 0.0005 ConclusionOverall, the E method was the most efficient method in the extraction of DNA from fungal cultures compared to the other methods considering time, cost, technical expertise, and instrumentation. Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays

Impact of DNA extraction methods on 16S rRNA-based profiling of bacterial communities in cheese

Numerous factors associated with sample preparation, DNA extraction, primer choice, sequencing platform and data analysis can affect the accuracy of 16S rRNA sequencing results. The DNA extraction method is considered critical for the success of sequencing as it can be the source of considerable variations in the analysis of the microbiome. In this study, the impact of various DNA extraction methods on the results of analysis of bacterial communities in cheese was evaluated. DNA was isolated from Mozzarella as a model cheese using optimized bead-based homogenization followed by different extraction procedures. Five commercial kits and two open-formula DNA extraction protocols were evaluated for amplicon sequencing of a 16S rRNA fragment of ~1460 bp. In addition, model cheese samples artificially contaminated by defined concentrations of Listeria monocytogenes and Escherichia coli, as representatives of Gram positive and Gram negative bacteria, were analysed. Six out of seven DNA extraction procedures were found to be able to provide amplifiable bacterial DNA suitable for 16S rRNA sequence analysis, but individual extraction procedures led to variable results. In particular, lysis supported with bead-beating led to a higher proportion of G+ bacteria in relative abundance profiles, probably because of the more efficient cell wall disruption. Artificially added bacterial species were reliably detected with a quantitative response. The results demonstrated a risk in comparing the data on bacterial communities in cheese when different DNA extraction protocols are used and highlighted the need to choose a standardized approach when comparison across multiple sequencing runs is required.

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood.

The molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories.

Comparison of different DNA extraction procedures for Eimeria oocysts

A study was carried out to compare the efficacy of different DNA extraction procedures for Eimerian oocysts. A total of 753 litter and fecal samples and intestines were collected from desi poultry farms and slaughter houses in Bengaluru, Karnataka. Out of five different methods of DNA extraction procedures the glass bead method was found to be the best method. Since the above method involves breaking of the oocysts and sporocysts walls and dissolving the sporozoites membrane. This greatly simplifies the currently used DNA extraction procedures for Eimeria species. The liquid nitrogen method was also found to be good but was more critical and time consuming. Other methods such as sodium hypochlorite, direct and sonication has failed to yield DNA from Eimeria oocysts. The present study indicates that glass beads method yielded better quality of DNA for molecular analysis and it is suitable for both large and small number (˂1x103) of oocysts in a sample. Hence, can be used as a routine procedure for DNA extraction for Eimerian oocysts.

Looking for Hidden Enemies of Metabarcoding: Species Composition, Habitat and Management Can Strongly Influence DNA Extraction while Examining Grassland Communities.

Despite the raising preoccupation, the critical question of how the plant community is composed belowground still remains unresolved, particularly for the conservation priority types of vegetation. The usefulness of metabarcoding analysis of the belowground parts of the plant community is subjected to a considerable bias, that often impedes detection of all species in a sample due to insufficient DNA quality or quantity. In the presented study we have attempted to find environmental factors that determine the amount and quality of DNA extracted from total plant tissue from above- and belowground samples (1000 and 10,000 cm2). We analyzed the influence of land use intensity, soil properties, species composition, and season on DNA extraction. The most important factors for DNA quality were vegetation type, soil conductometry (EC), and soil pH for the belowground samples. The species that significantly decreased the DNA quality were Calamagrostis epigejos, Coronilla varia, and Holcus lanatus. For the aboveground part of the vegetation, the season, management intensity, and certain species—with the most prominent being Centaurea rhenana and Cirsium canum—have the highest influence. Additionally, we found that sample size, soil granulation, MgO, organic C, K2O, and total soil N content are important for DNA extraction effectiveness. Both low EC and pH reduce significantly the yield and quality of DNA. Identifying the potential inhibitors of DNA isolation and predicting difficulties of sampling the vegetation plots for metabarcoding analysis will help to optimize the universal, low-cost multi-stage DNA extraction procedure in molecular ecology studies.

Comparative analysis of DNA extraction processes for DNA-based identification from putrefied bodies in forensic routine work

Identification of putrefied bodies is an important and common task in forensic routine. Usually, the identification of deceased is done by visual recognition, fingerprints, physical distinguishing marks (e.g. tattoos, scars and surgical implants) and/or dental examination. However, if morphologic characteristics are not recognizable, due to advanced putrefaction of the corpse, or recent medical records are not available, the DNA-based identification is favored.Thus, in order to find another reliable procedure for DNA extraction of putrefied samples regarding tissue selection, costs and time, two commercial forensic kits were compared: DNeasy® Blood & Tissue kit and SwabSolution™ kit. Both kits were used for DNA extraction from five soft tissues (brain, aorta, liver, kidney and psoas major muscle) and nails (finger- and toenail) obtained during the medicolegal autopsy of 20 putrefied corpses. DNeasy® Blood & Tissue kit was by quantitative comparison mostly superior to SwabSolution™ kit: it yielded more DNA of better quality (i.e. less degraded and inhibited). However, the qualitative results (DNA profiles) of both extraction procedures were similar. Among the analyzed tissue types, nails were proved as the most suitable for DNA-based identification of putrefied bodies.

Otitis media with effusion: The role of Helicobacter Pylori in its pathogenesis

BackgroundOtitis Media with Effusion (OME) is the most common disease of the middle ear. Different factors play a role in its pathogenesis, such as viral and bacterial infections, allergy, morphological and functional changes of nasal passage, Eustachian Tube (ET), and cleft palate. This study aims to investigate the Helicobacter Pylori presence in middle ear effusions from patients with OME through RT-PCR and compare our results with results from other published articles. MethodsThe study was carried out from October 2007 to February 2009, in the Department of Otorhinolaryngology of SS. Annunziata Hospital, Chieti, Italy. 132 consecutive patients with OME were included in the study. Fluid in the middle ear was assessed for the presence of Helicobacter Pylori through RT-PCR. Results132 consecutive patients with OME were included in the study. The patients were between ages 8 and 78 (median 50); 62 were males (47%), 70 were females (53%), and 53 patients had bilateral OME (40%). 185 samples were collected from 132 patients. Of the 185 samples taken from the ear, 21 (11.35%) were not adequate for the correct execution of the DNA extraction procedure. The remaining 167 samples, subjected to RT-PCR, did not show in any case an increase in fluorescence linked to the FAM fluorophore, thus demonstrating the complete absence of Helicobacter Pylori. ConclusionBased on the results obtained, we can affirm that although a third of the cases of OME is correlated to the presence of reflux, Helicobacter Pylori does not seem to play any role in the pathophysiology of OME as it cannot be found in endo-tympanic exudate.

Molecular Methods for the Detection of Toxoplasma gondii Oocysts in Fresh Produce: An Extensive Review.

Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure.

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth. Key points Bones and teeth often represent the only sources of DNA for identifying human remains. The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors. This study focuses on modifications to the previously available Qiagen-based protocols. The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols. The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

Optimized protocol for DNA extraction from ancient skeletal human remains using Chelex-100

PCR-based analysis of skeletonized human remains is a common aspect in both forensic human identification as well as Ancient DNA research. In this, both areas not merely utilize very similar methodology, but also share the same problems regarding quantity and quality of recovered DNA and presence of inhibitory substances in samples from excavated remains. To enable amplification based analysis of the remains, development of optimized DNA extraction procedures is thus a critical factor in both areas.The study here presents an optimized protocol for DNA extraction from ancient skeletonized remains using Chelex-100, which proved to be effective in yielding amplifiable extracts from sample material excavated after centuries in a soil environment, which consequently have high inhibitor content and overall limited DNA preservation. Success of the optimization strategies utilized is shown in significantly improved amplification outcomes compared to the predecessor method

Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.

Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.

Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study.

We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.

A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae)

Grape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5′-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT “HS6”, for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.

DNA Extraction with TRIzol Reagent Using a Silica Column.

TRIzol is a monophasic solution of phenol and guanidine isothiocyanate used for the extraction of RNA, DNA and proteins from tissues or cells. However, few studies have described its application to DNA extraction due to its time-consuming procedure. We present a TRIzol-modified method of extracting DNA from tissues using the TRIzol reagent and a silica column, which requires only one-third of the time required for the classic extraction procedure. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the DNA extracted using the TRIzol-modified method is ideal and equal to that obtained by the classic method and commercial DNAiso methods. The DNA extracted by the TRIzol-modified method had the same performance in a restriction enzyme digestion and quantitative PCR as that extracted using the classic method. Using the TRIzol-modified method saves time, simplifies the DNA extraction procedure, and facilitates various molecular biology assays.

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Procedures for DNA Extraction from Opium Poppy (Papaver somniferum L.) and Poppy Seed-Containing Products.

Several commonly used extraction procedures and commercial kits were compared for extraction of DNA from opium poppy (Papaver somniferum L.) seeds, ground seeds, pollen grains, poppy seed filling from a bakery product, and poppy oil. The newly developed extraction protocol was much simpler, reduced the cost and time required for DNA extraction from the native and ground seeds, and pollen grains. The quality of extracted DNA by newly developed protocol was better or comparable to the most efficient ones. After being extended by a simple purification step on a silica membrane column, the newly developed protocol was also very effective in extracting of poppy DNA from poppy seed filling. DNA extracted from this poppy matrix was amplifiable by PCR analysis. DNA extracted from cold-pressed poppy oil and suitable for amplifications was obtained only by methods developed previously for olive oil. Extracted poppy DNA from all tested matrices was analysed by PCR using primers flanking a microsatellite locus (156 bp) and two different fragments of the reference tubulin gene (553 bp and 96 bp). The long fragment of the reference gene was amplified in DNA extracted from native seeds, ground seeds, and pollen grains. Poppy DNA extracted from the filling of bakery product was confirmed only by amplification of short fragments (96 bp and 156 bp). DNA extracted from cold-pressed poppy oil was determined also only by amplification of these two short fragments.

New multiplex conventional PCR and quadruplex real-time PCR assays for one-tube detection of Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis in Citrus: development and validation.

Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100pgμL-1 for P. citricarpa, E. fawcettii, and E. australis and 10pgμL-1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. KEY POINTS: • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

Learning machine approach reveals microbial signatures of diet and sex in dog.

The characterization of the microbial population of many niches of the organism, as the gastrointestinal tract, is now possible thanks to the use of high-throughput DNA sequencing technique. Several studies in the companion animals field already investigated faecal microbiome in healthy or affected subjects, although the methodologies used in the different laboratories and the limited number of animals recruited in each experiment does not allow a straight comparison among published results. In the present study, we report data collected from several in house researches carried out in healthy dogs, with the aim to seek for a variability of microbial taxa in the faeces, caused by factors such as diet and sex. The database contains 340 samples from 132 dogs, collected serially during dietary intervention studies. The procedure of samples collection, storage, DNA extraction and sequencing, bioinformatic and statistical analysis followed a standardized pipeline. Microbial profiles of faecal samples have been analyzed applying dimensional reduction discriminant analysis followed by random forest analysis to the relative abundances of genera in the feces as variables. The results supported the responsiveness of microbiota at a genera taxonomic level to dietary factor and allowed to cluster dogs according this factor with high accuracy. Also sex factor clustered dogs, with castrated males and spayed females forming a separated group in comparison to intact dogs, strengthening the hypothesis of a bidirectional interaction between microbiota and endocrine status of the host. The findings of the present analysis are promising for a better comprehension of the mechanisms that regulate the connection of the microorganisms living the gastrointestinal tract with the diet and the host. This preliminary study deserves further investigation for the identification of the factors affecting faecal microbiome in dogs.

Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR protocols for mouse studies.

Droplet digital PCR (ddPCR) is a recent method developed for the quantification of nucleic acids sequences. It is an evolution of PCR methodology incorporating two principal differences: a PCR reaction is performed in thousands of water-oil emulsion droplets and fluorescence is measured at the end of PCR amplification. It leads to the precise and reproducible quantification of DNA and RNA sequences. Here, we present quantitative methods for DNA and RNA analysis using Bio-Rad QX100 or QX200 systems, respectively. The aim of these methods is to provide useful molecular tools for validating genetically altered animal models such as those subject to CRISPR/Cas9 genome editing, as well for expression or CNV studies. A standard procedure for simultaneous DNA and RNA extraction adapted for mouse organs is also described. These methods were initially designed for mouse studies but also work for samples from other species like rat or human. In our lab, thousands of samples and hundreds of target genes from genetically altered lines were examined using these methods. This large dataset was analyzed to evaluate technical optimizations and limitations. Finally, we propose additional recommendations to be included in dMIQE (Minimum information for publication of quantitative digital PCR experiments) guidelines when using ddPCR instruments.