Choice of gDNA isolation method has a significant impact on average murine Telomere Length estimates.

Telomere Length (TL) and integrity is significantly associated with age-related disease, multiple genetic and environmental factors. We observe mouse genomic DNA (gDNA) isolation methods to have a significant impact on average TL estimates. The canonical qPCR method does not measure TL directly but via the ratio of telomere repeats to a single copy gene (SCG) generating a T/S ratio. We use a monochromatic-multiplex-qPCR (mmqPCR) method which multiplexes the PCR and enables quantification of the target and the single copy gene within the same qPCR reaction. We demonstrate that TL measurements, from murine gDNA, isolated via Spin Columns (SC) and Magnetic Beads (MB), generate significantly smaller T/S ratios compared to gDNA isolated via traditional phenol/chloroform methods. The former methods may impede correct TL estimation by producing non representative fragment sets and reducing qPCR efficacy. This work highlights discrepancies in TL measurements due to different extraction techniques. We recommend the use of gDNA isolation methods that are shown to preserve DNA length and integrity, such as phenol/chloroform isolation. We propose that widely used high throughput DNA isolation methodologies can create spurious associations within a sample set, thus creating misleading data. We suggest that published TL associations should be revisited in the light of these data.

Two-step method for rapid isolation of genomic DNA and validation of R81T insecticide resistance mutation in Myzus persicae.

Isolation and amplification of nucleic acid (DNA) is considered a vital and potent instrument in molecular biological research. However, its functioning outside of a laboratory setting is difficult because of complex procedures that demand expert personnel and expensive equipment in addition to the fulfillment of several additional requirements. DNA isolation from minute insects is sometimes difficult, making diagnostic and genotyping procedures problematic. Thus, the current work offers a high-throughput, cost-effective, straightforward, and faster approach for isolating DNA from the aphid Myzus persicae. Intriguingly, two-step DNA extraction process yielded a high yield of extremely pure genomic DNA and required only 10 s to complete. PCR investigation aiming at amplifying the non-synonymous R81T region on the loop D site of the nAChR gene of M. persicae was subsequently utilized to successfully validate the recovered DNA. Moreover, the proposed method was compared in terms of yield and purity with conventionally used DNA isolation methods including, phenol:chloroform, salt out, and commercially available kits. In conclusion, this newly developed method would enable researchers to quickly process many biological samples used to analyze genetic diversity, mutant screening, and large spectrum diagnosis both in laboratory and field conditions.

Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma.

BACKGROUNDS: Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma. AIMS: First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays. MATERIALS & METHODS: For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. RESULTS: In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10-3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. DISCUSSION: A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. CONCLUSION: Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.

Phenol/Chloroform-Free TiO2-Based miRNA Extraction from Cell Lysate.

While microRNAs are considered as excellent biomarkers of various diseases, there are still several remaining challenges regarding their isolation. In this study, we aimed to design a novel RNA isolation method that would help to overcome those challenges. Therefore, we present a novel phenol/chloroform-free, low-cost method for miRNA extraction. Within this method, RNA is extracted from cell lysate with an isopropanol/water/NaCl system, followed by solid-phase extraction using TiO2 microspheres to effectively separate short RNAs from long RNA molecules. We also demonstrated the pH-dependent selectivity of TiO2 microspheres towards different sizes of RNA. We were able to regulate the size range of extracted RNAs with simple adjustments in binding conditions used during the solid-phase extraction.

UPA-Seq-Based Search Method for Functional lncRNA Candidates.

Long noncoding RNAs (lncRNAs) constitute a large fraction of the transcriptome in mammals, and recent studies have revealed important functions of lncRNAs in a variety of biological processes. However, the fraction of lncRNAs that have been functionally validated is small, and only sequence and expression information are available for most lncRNAs. Here, we describe the procedures for UV-phenol aqueous-phase RNA sequencing (UPA-seq), a method for searching for functional lncRNA candidates among whole genomes based on the assumption that functional lncRNAs exert their functions through associations with proteins.

Evaluation of RNA Isolation Methods in Human Adipose Tissue.

OBJECTIVE: Research has shown that RNA extraction from adipose tissue (AT) is challenging because of high lipid content and low RNA quantity. We compared a traditional RNA extraction with a column-based method in human AT to evaluate RNA quantity and quality. MATERIALS AND METHODS: Human subcutaneous AT (n = 9) was collected through needle biopsy, and RNA was extracted using the phenol-chloroform traditional method and the RNeasy Lipid Tissue Mini Kit column-based method. The RNA quantity, quality, integrity, and expression of key AT genes were assessed. RESULTS: We found that the RNA quantity and integrity were reduced by 40% and 15-20%, respectively, using the column-based method compared to the traditional method, but the findings were not statistically significant. The column-based method showed a higher 260/280 ratio (~2.0) compared to the traditional method (~1.8) (P <.05), suggesting lower amounts of contaminants. The expression of AT genes was comparable between methods. CONCLUSION: The traditional extraction method provides adequate RNA yield and integrity compared to the column-based method, which is an advantage when AT specimens are small.

Retrieving high-quality genomic DNA from formalin-fixed paraffin-embedded tissues for multiple molecular analyses.

Formalin-fixed paraffin-embedded (FFPE) tissues represent the biggest source of archival materials for molecular biology research and pathology investigations. Nevertheless, fixation by formalin may cause denaturation and modification of macromolecules constraining DNA quality and its downstream applications. In this study, we developed a fast, simple, and cost-effective phenol/chloroform-based protocol for the extraction of high-quality DNA from 101 FFPE colorectal cancer tissue blocks that can be used in multiple molecular studies. DNA samples extracted using this phenol/chloroform protocol and the QIAamp DNA FFPE Tissue kit were evaluated for the quantity, quality, and amplificability. Spectrophotometer analyses revealed significantly higher quality and quantity of DNA samples obtained with the phenol/chloroform protocol as compared to those of the QIAamp DNA FFPE Tissue kit. In addition, the amplificability of these samples as assessed by conventional and multiplex polymerase chain reaction (PCR), followed by sequencing and fragment analyses presented an absolute success rate. Additionally, it is able to amplify a DNA fragment of 725 base-pairs at an adequate amount for downstream applications. This fast, simple, and cost-effective method may facilitate the molecular analyses of a large number of FFPE specimens that best suits the needs of the overall study design in terms of the quality and quantity of the extracted DNA.

Forensic human identification: Investigation into tooth morphotype and DNA extraction methods from teeth.

When a body is decomposed, hard tissues such as teeth may provide the only DNA source for human identification. There is currently no consensus as to the best DNA extraction method, and there is a lack of empirical data regarding tooth morphotype and condition that may impact DNA recovery. Therefore, this study sought to investigate which variables significantly improved DNA concentration, integrity and profiling success. A total of 52 human teeth were assessed, representing all tooth morphotypes from three deceased individuals. DNA was extracted using both the QIAamp® DNA Investigator Kit and the phenol-chloroform method. DNA concentration and degradation index were assessed using real time PCR, prior to conventional DNA profiling. Contrary to international guidelines promoting the use of molars, DNA profiling from molars was the least successful, with premolars, followed by canines, performing the best. The presence of fillings reduced the DNA quantity and quality obtained and may explain the poor performance of molars. DNA from the maxillae were significantly less degraded when the QIAamp® was used, although this did not influence DNA profiling success. A significant increase in DNA concentration, integrity and profiling success was observed in diseased teeth (periodontitis) compared to those without disease. This may be due to increased white blood cell presence at the site. There was no significant difference in DNA profiling success between the two DNA extraction methods. However, different teeth yielded failed DNA profiles for each extraction method, suggesting that repeated attempts, using alternative DNA extraction methods, is recommended. The recovery of additional DNA profiling information from degraded samples may help to ultimately reduce the burden of unidentified human remains.

High-yield skeletal muscle protein recovery from TRIzol after RNA and DNA extraction.

Extraction of DNA, RNA and protein from the same sample would allow for direct comparison of genomic, transcriptomic and proteomic information. Commercially available kits exhibit poor protein yield and the TRIzol® reagent produces a protein pellet that is extremely difficult to solubilize. In response to these limitations, this study presents an optimized method for the extraction of protein from the organic phase of TRIzol that allows for higher yield recovery of skeletal muscle protein compared with direct homogenization in a common protein lysis buffer. The presented method is inexpensive, simple and fast, requires no additional treatment of the protein pellet for dissolution, and is compatible with downstream western blot applications.

The Single-Step Method of RNA Purification Applied to Leptospira.

Establishing a rapid method to obtain pure and intact RNA molecules has revolutionized the field of RNA biology, enabling laboratories to routinely perform RNA analysis such as Northern blot, reverse transcriptase quantitative PCR, and RNA sequencing. Here, we describe an application of the effective single-step method of RNA extraction (or guanidinium thiocyanate-phenol-chloroform extraction) applied to Leptospira species. This method is based on the powerful ability of guanidinium thiocyanate to inactivate RNases and on the different solubilities of RNA and DNA in acidic phenol. This method allows one to reproducibly obtain total RNAs with high yield and integrity, as determined by capillary electrophoresis, suitable for the RNA sequencing technology.

Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria.

Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5'-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

Isolation of High-Quality RNA from Pichia pastoris.

Analysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastoris requires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition. We therefore set out to compare the efficacy of two different RNA isolation methods when applied to P. pastoris: (i) phenol/chloroform extraction and (ii) silica spin-column absorption. We compared the yield, integrity, and purity of the resulting isolated RNA from the two methods (using two different types of commercial columns for silica spin-column absorption) and further optimized them through variations in the pretreatment steps. We also assessed two different methods of cell lysis: enzyme catalytic disruption using lyticase and mechanical disruption using acid-washed glass-beads in a TissueLyser. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: RNA isolation with phenol/chloroform extraction: monophasic lysis reagent Alternate Protocol 1: RNA isolation with silica-spin column absorption: High Pure RNA Isolation Kit (Roche Life Science) Alternate Protocol 2: RNA isolation with silica-spin column absorption: RNeasy Mini Kit (Qiagen).

Comparison of two DNA extraction methods widely used in aquatic microbial ecology.

In recent years, the rapid advances of culture-independent methods and new molecular tools have revolutionized our understanding of microbial biodiversity and ecological functions. DNA extraction from microbial communities is a critical step in this process and several methods have been proposed and used, but the influence of the extraction method on the outcome and ultimately on ecological inferences from the results is not yet precisely determined. Here, we compared two of the most commonly used extraction methods in aquatic microbial ecology, and investigated whether the two methods yielded comparable results for community ecology analyses. We extracted DNA from 15 different shallow lakes with phenol:chloroform, a classical and widely used extraction method, and with the PowerSoil DNA isolation Kit, often suggested as the standard DNA extraction method, with some adaptations for aquatic environments. We found that although only 5% of all OTUs showed significant differences in pairwise comparisons (using the 15 lakes as replicates), these OTUs accounted for >35% (on average) of the relative abundance. Diversity and richness did not differ significantly between the two extraction methods, but the beta-dispersion of the communities indicated that the organic extraction yielded more homogeneous communities, while the kit extraction generated variability. Consequently, we conclude that despite the small number of OTUs with significant differences, their impact on the community composition obtained was not negligible, and therefore the results from these two extraction methods were not comparable.

Heat Inactivation of Coccidioides posadasii and Coccidioides immitis for Use in Lower Biosafety Containment.

INTRODUCTION: The difficulty involved in obtaining sufficient intact genomic deoxyribonucleic acid (DNA) from Coccidioides spp for downstream applications using published protocols prompted the exploration of inactivating mycelia and arthroconidia using heat under biosafety level 3 containment. This was followed by optimizing DNA extraction from mycelia using various methods at lower containment. METHODS: Various exposure times and temperatures were examined to identify an effective heat inactivation procedure for arthroconidia and mycelia from both C immitis and C posadasii. Heat inactivation of mycelia was followed by DNA extraction using 2 commercially available kits, as well as a phenol:chloroform-based extraction procedure to determine DNA integrity and quantity among extraction methods using both live and heat-inactivated mycelia. RESULTS: Ten-minute and 30-minute exposure times at 80°C were sufficient to inactivate Coccidioides spp arthroconidia and mycelia, respectively. DNA yield between live versus heat-inactivated mycelia was similar for each extraction procedure. However, DNA obtained using phenol:chloroform was of higher quantity and integrity compared with DNA obtained using the commercially available kits, which was highly fragmented. CONCLUSION: The ability to heat-inactivate Coccidioides cultures for processing at a lower level of containment greatly increased the efficiency of DNA extractions. Therefore, this is an ideal method for obtaining Coccidioides spp DNA and inactivated arthroconidia.

UPA-seq: prediction of functional lncRNAs using differential sensitivity to UV crosslinking.

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-seq). As expected, the numbers of UPA-seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

Optimization of phenol-chloroform RNA extraction.

Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. •Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.

Planarian High Molecular Weight DNA Isolation by Spooling.

High-quality large molecular weight genomic DNA is important for genomic studies. Most commercial available genomic DNA purification kits have failed to generate high molecular weight DNA of sufficient quality from planarians. Here, we describe a simple and efficient genomic DNA isolation method, which has worked for several different planarian species, including Schmidtea mediterranea. This phenol-chloroform based method can be used to obtain genomic DNA of up to 150 kb and can be used for bacterial artificial chromosome (BAC) library construction, next-generation sequencing and PCR cloning.

DNA extraction from benthic Cyanobacteria: comparative assessment and optimization.

AIMS: Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. METHODS AND RESULTS: The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. CONCLUSIONS: Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing.

Assessing DNA recovery from chewing gum.

The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Restriction Endonuclease Analysis Typing of Clostridium difficile Isolates.

Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.