Catalogue Search | MBRL
Are you sure you want to remove the book from the shelf?
{{itemTitle}}
447
result(s) for
"Cole, Kenneth"
Sort by:
Standards for Cell Line Authentication and Beyond
Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines.
Journal Article
Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR
Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.
Journal Article
Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine
Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use.
We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (
c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement.
Concentration values for samples of
G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively).
This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.
Journal Article
Middle Holocene flora and fauna from a ringtail (Bassariscus, Carnivora) den, western Grand Canyon, Arizona
Dietary remains recovered from Bassariscus (ringtail) midden deposits of middle Holocene age in the Weeping Cliffs, lower Grand Canyon, Arizona, indicate a diverse ringtail diet of plants and small vertebrates. Remains of fruits and seeds predominated among the plant parts, especially of hackberry, several cacti, and groundcherry. Highly fragmented remains of small vertebrates included anurans, lizards, snakes, bats, and rodents. Only one recovered vertebrate specimen exceeded 5 mm in length, necessitating meticulous morphological comparisons for taxonomic identifications, which were usually limited to generic level. Two radioisotopic dates on the middens range from ca. 7300 to 7900 cal yr BP. Vertebrate remains from the middens provide the first Quaternary records of amphibians (including Anaxyrus and Hyla/ Dryophytes) and a bat (Nyctinomops macrotis) from the Grand Canyon. Rare, fragmentary specimens provide one of only two fossil records of Dipsosaurus in Arizona.
Journal Article
Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR)
Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment.
Journal Article
Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells
Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.
Journal Article
Understanding medical students’ knowledge, attitudes, and preparatory needs for future prescribing of long-acting injectable pre-exposure prophylaxis
Injectable pre-exposure prophylaxis (PrEP) offers a novel approach to Human Immunodeficiency Virus (HIV) prevention and may increase accessibility for people vulnerable to HIV acquisition. However, effective implementation will require that healthcare providers are aware and knowledgeable of injectable PrEP. One avenue to increasing awareness and knowledge among healthcare providers is to educate future providers during their medical education. From June to August 2022, we conducted in-depth, semi-structured interviews with medical students (
N
= 32) to better understand their perceptions and attitudes toward injectable PrEP, as well as their preparatory needs for prescribing injectable PrEP in the future. Thematic analyses highlighted two primary domains: (1) Perceptions of injectable PrEP and (2) Preparatory educational needs for prescribing injectable PrEP in the future. Expansive themes centered around low injectable PrEP awareness and knowledge, concerns about the accessibility of injectable PrEP, patient-centered benefits of injectable PrEP, interest in developing patient-provider communication skills, desire for comprehensive education about injectable PrEP and its prescribing practices, and preferences for enhanced learning about injectable PrEP. Results from this study suggest medical students desire specific, patient-centred educational opportunities to prescribe injectable PrEP in the future. Understanding these preparatory needs will assist with developing innovative training opportunities centred on HIV prevention and treatment.
Journal Article
Vegetation Response to Early Holocene Warming as an Analog for Current and Future Changes
Temperatures in southwestern North America are projected to increase 3.5-4 °C over the next 60-90 years. This will precipitate ecological shifts as the ranges of species change in response to new climates. During this shift, rapid-colonizing species should increase, whereas slow-colonizing species will at first decrease, but eventually become reestablished in their new range. This successional process has been estimated to require from 100 to over 300 years in small areas, under a stable climate, with a nearby seed source. How much longer will it require on a continental scale, under a changing climate, without a nearby seed source? I considered this question through an examination of the response of fossil plant assemblages from the Grand Canyon, Arizona, to the most recent rapid warming of similar magnitude that occurred at the start of the Holocene, 11,700 years ago. At that time, temperatures in southwestern North America increased about 4 °C over less than a century. Grand Canyon plant species responded at different rates to this warming climate. Early-successional species rapidly increased, whereas late-successional species decreased. This shift persisted throughout the next 2700 years. I found two earlier, less-extreme species shifts following rapid warming events around 14,700 and 16,800 years ago. Late-successional species predominated only after 4000 years or more of relatively stable temperature. These results suggest the potential magnitude, duration, and nature of future ecological changes and have implications for conservation plans, especially those incorporating equilibrium assumptions or reconstituting past conditions. When these concepts are extended to include the most rapid early-successional colonizers, they imply that the recent increases in invasive exotics may be only the most noticeable part of a new resurgence of early-successional vegetation. Additionally, my results challenge the reliability of models of future vegetation and carbon balance that project conditions on the basis of assumptions of equilibrium within only a century.
Journal Article
