Assessing neophyte response to daily disposable silicone hydrogel contact lenses: A randomised clinical trial investigation over one month.

OBJECTIVE: This randomised clinical trial assessed the impact on symptoms, tear film dynamics and ocular surface integrity of daily disposable silicone-hydrogel contact lenses (CLs) over a month, paying special attention to lid wiper epitheliopathy (LWE) and its implications for CL discomfort. METHODS: Neophyte CL wearers (n = 44, 21.09 ± 5.00 years old) were randomly assigned to either the experimental (n = 24) or control group (n = 20). Participants assigned to the experimental group were required to wear daily disposable CLs for 1 month for at least 8 h/day and 6 days/week. All participants were healthy subjects (no history of ocular surgery or active ocular disease) with spherical refractive errors between -8.00 and +5.00 D and cylindrical power <0.75 D. At the baseline and 1-month sessions, the Dry Eye Questionnaire 5 (DEQ-5) was completed, together with the measurement of tear film osmolarity with the TearLab osmometer, tear meniscus height (TMH) and lipid layer pattern (LLP) using a slit-lamp with Tearscope Plus attached, fluorescein break-up time (FBUT), maximum blink interval (MBI), corneal staining with fluorescein under cobalt blue light and LWE with lissamine green under slit lamp and halogen white light. RESULTS: At the baseline session, LWE showed a negative correlation with DEQ-5 (r = -0.37, p = 0.02). Significant differences in FBUT and LWE (p = 0.04) and a positive correlation between LWE and DEQ-5 (r = 0.49, p = 0.007) were observed at 1 month. Intrasession analysis at 1 month showed significant differences between the experimental and control groups in DEQ-5, FBUT and LWE (all p ≤ 0.02). Intersession analysis in the experimental group showed variations in DEQ-5, FBUT and LWE (all p ≤ 0.02) but no significant variation in the control group (all p ≥ 0.11). CONCLUSION: The presence of LWE was significantly correlated with higher symptom values in the DEQ-5. Also, participants in the experimental group presented higher values of LWE after 1 month of CL wear, in comparison with the control group.

ctDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV+ oropharyngeal cancer.

BACKGROUNDTransrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.MethodsUsing whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.ResultsTR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.ConclusionOur data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.FundingNIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.

Unveiling a Technosol-based remediation approach for enhancing plant growth in an iron-rich acidic mine soil from the Rio Tinto Mars analog site.

This paper explores the potential of Technosols made from non-hazardous industrial wastes as a sustainable solution for highly acidic iron-rich soils at the Rio Tinto mining site (Spain), a terrestrial Mars analog. These mine soils exhibit extreme acidity (pHH2O = 2.1-3.0), low nutrient availability (non-acid cation saturation < 20 %), and high levels of Pb (3420 mg kg-1), Cu (504 mg kg-1), Zn (415 mg kg-1), and As (319 mg kg-1), hindering plant growth and ecosystem restoration. To address these challenges, the study systematically analyzed selected waste materials, formulated them into Technosols, and conducted a four-month pot trial to evaluate the growth of Brassica juncea under greenhouse conditions. Technosols were tailored by adding varying weight percentages of waste amendments into the mine Technosol, specifically 10 %, 25 %, and 50 %. The waste amendments comprised a blend of organic waste (water clarification sludge, WCS) and inorganic wastes (white steel slag, WSS; and furnace iron slag, FIS). The formulations included: (T0) exclusively mine Technosol (control); (T1) 60 % WCS + 40 % WSS; (T2) 60 % WCS + 40 % FIS; and (T3) 50 % WCS + 16.66 % WSS + 33.33 % FIS. The analyses covered leachate quality, soil pore water chemistry, and plant response (germination and survival rates, plant height, and leaf number). Results revealed a significant reduction in leachable contaminant concentrations, with Pb (26.16 mg kg-1), Zn (4.94 mg kg-1), and Cu (2.29 mg kg-1) dropping to negligible levels and shifting towards less toxic species. These changes improved soil conditions, promoting seed germination and seedling growth. Among the formulations tested, Technosol T1 showed promise in overcoming mine soil limitations, enhancing plant adaptation, buffering against acidification, and stabilizing contaminants through precipitation and adsorption mechanisms. The paper stresses the importance of tailoring waste amendments to specific soil conditions, and highlights the broader implications of the Technosol approach, such as waste valorization, soil stabilization, and insights for Brassica juncea growth in extreme environments, including Martian soil simulants.

Tear film osmolarity variation between weeks in healthy and dry eye disease subjects / Variação semanal da osmolaridade do filme lacrimal em indivíduos saudáveis e com síndrome do olho seco

ABSTRACT Purpose: The possible variability in diagnostic test results is a statistical feature of dry eye disease patients. The clinician should consider tear film variations over time since the timing of tear film measurements is important for proper diagnosis. The purpose of the present study was to analyze the inter-week variation of osmolarity measurement in healthy and dry eye disease participants. Methods: Based on the Dry Eye Workshop II (DEWS-II) diagnostic methodology report criteria, a battery of tests (Ocular Surface Disease Index [OSDI] questionnaire, breakup time, and corneal staining) was administered to rule out the presence of dry eye disease. A total of 40 qualified volunteers were recruited into two groups: with only 20 healthy and 20 dry eye disease participants. The inter-week variation of osmolarity in the two groups was measured using a TearLab osmometer in two sessions one-week apart. The differences between the results were calculated. Results: There were no significant differences in osmolarity between the two sessions for either the healthy (paired t-test; p=0.085) or dry eye disease (paired t-test; p=0.093) participants. Moreover, there was no significant correlation between the means and differences in either session on healthy (Pearson correlation: r=0.020; p=0.935) or dry eye disease (Pearson correlation: r=-0.022; p=0.928) participants. In session 1, there was a significant difference in osmolarity values between groups (unpaired t-test; p=0.001), but no difference was found in session 2 (unpaired t-test; p=0.292). Conclusions: The present study discovered no inter-week variation in the tear film osmolarity of healthy and dry eye disease participants classified based on the DEWS-II criteria.

RESUMO Objetivo: A possível variabilidade nos resultados de testes diagnósticos é uma característica estatística dos pacientes com síndrome do olho seco. O médico deve considerar as variações do filme lacrimal ao longo do tempo, pois o momento em que o filme lacrimal é medido pode ser crítico para o diagnóstico adequado. O objetivo deste estudo foi analisar a variação semanal da osmolaridade do filme lacrimal em participantes saudáveis e em outros com síndrome do olho seco. Métodos: Com base nos critérios da metodologia de diagnóstico do relatório da Dry Eye Workshop II (DEWSII), foi aplicada uma bateria de testes (questionário do índice de doença da superfície ocular [OSDI], tempo de ruptura do filme lacrimal e coloração da córnea) para descartar a presença de síndrome do olho seco. Um total de 40 voluntários qualificados foi recrutado e distribuído em dois grupos de 20 participantes saudáveis e 20 participantes com síndrome do olho seco. A variação da osmolaridade entre semanas foi medida com um osmômetro TearLab em duas sessões com uma semana de intervalo nos dois grupos. As diferenças entre os resultados foram então calculadas. Resultados: Não foram encontradas diferenças significativas na osmolaridade entre as medidas obtidas nas duas sessões, nem no grupo de participantes saudáveis (teste de t pareado; p=0,085), nem no de participantes com síndrome do olho seco (teste de t pareado; p=0,093). Não foi detectada nenhuma correlação significativa entre as médias e diferenças entre as duas sessões entre participantes saudáveis (correlação de Pearson: r=0,020, p=0,935) e aqueles com síndrome do olho seco (correlação Pearson: r=-0,022, p=0,928). Foi encontrada uma diferença significativa nos valores de osmolaridade entre os dois grupos na primeira sessão (teste de t não pareado; p=0,001), mas nenhuma diferença foi encontrada na segunda sessão (teste de t não pareado; p=0,292). Conclusões: O presente estudo não encontrou variação entre semanas consecutivas na osmolaridade do filme lacrimal em participantes saudáveis e com síndrome do olho seco, classificados com base nos critérios do DEWSII.

Cysteamine Eye Drops in Hyaluronic Acid Packaged in Innovative Single-Dose Systems, Part II: Long-Term Stability and Clinical Ocular Biopermanence.

BACKGROUND: Cystinosis is a rare genetic disorder characterized by the accumulation of cystine crystals in several tissues and organs causing, among others, severe eye symptoms. The high instability of cysteamine eye drops makes it difficult to develop formulations with an acceptable shelf life to be prepared in hospital pharmacy departments. Previously, a new compounded formulation of cysteamine eye drops in hyaluronic acid (HA) packaged in innovative single-dose systems was developed. METHODS: Long-term stability at -20 °C of this formulation was studied considering the content of cysteamine, pH, osmolality, viscosity, and microbiological analysis. The oxygen permeability of single-dose containers was also studied and an ocular biopermanence study was conducted in healthy volunteers measuring lacrimal stability and volume parameters. RESULTS: Data confirm that cysteamine concentration remained above 90% for 120 days, all parameters remaining within the accepted range for ophthalmic formulations. The permeability of the containers was reduced over time, while ocular biopermanence was maintained despite the freezing process and storage time. CONCLUSIONS: 0.55% cysteamine hydrochloride formulation in HA and packaged in single-dose containers preserved at -20 °C is stable for 120 days protected from light, presenting high potential for its translation into clinical practice when commercial presentations are not available.

New insights into IL-6 family cytokines in metabolism, hepatology and gastroenterology.

IL-6 family cytokines are defined by the common use of the signal-transducing receptor chain glycoprotein 130 (gp130). Increasing evidence indicates that these cytokines are essential in the regulation of metabolic homeostasis as well as in the pathophysiology of multiple gastrointestinal and liver disorders, thus making them attractive therapeutic targets. Over the past few years, therapies modulating gp130 signalling have grown exponentially in several clinical settings including obesity, cancer and inflammatory bowel disease. A newly engineered gp130 cytokine, IC7Fc, has shown promising preclinical results for the treatment of type 2 diabetes, obesity and liver steatosis. Moreover, drugs that modulate gp130 signalling have shown promise in refractory inflammatory bowel disease in clinical trials. A deeper understanding of the main roles of the IL-6 family of cytokines during homeostatic and pathological conditions, their signalling pathways, sources of production and target cells will be crucial to the development of improved treatments. Here, we review the current state of the role of these cytokines in hepatology and gastroenterology and discuss the progress achieved in translating therapeutics targeting gp130 signalling into clinical practice.

Phospho-RNAseq Profiling of Extracellular mRNAs and lncRNAs.

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long noncoding RNA (lncRNA) studies are limited. We have recently reported that human plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA-seq protocols due to lack of 5'phosphate or presence of 3'phosphate. Here, we describe a modified protocol for preparation of small RNA libraries for next generation sequencing called "phospho-RNA-seq." This protocol has been optimized for use with low-input exRNA-containing samples, such as plasma or serum, and has modifications introduced to capture extracellular RNA with varied 5'and 3'ends.

Inter-eye osmolarity differences in patients with symptomatic and non-symptomatic dry eyes / Diferenças de osmolaridade entre olhos de pacientes com olhos secos sintomáticos e não sintomáticos

ABSTRACT Purpose: To analyze whether inter-eye osmo larity differences were related to dry eye symptomatology. Methods: A total of 135 participants were randomly recruited from those who visited in the Optometry Clinic of the Optometry Faculty (Universidade de Santiago de Compostela). In a single scheduled session after the recruitment, Ocular Surface Disease Index was filled out following the standard instructions and TearLab measurements were made in both the participants' eyes (10-15 min lapse). Osmolarity values were compared between the right and left eyes and the absolute inter-ocular difference (-OD-OS-) correlated with the Ocular Surface Disease Index score for the whole sample. Based on the Ocular Surface Disease Index score, the sample was divided into four symptomatic subgroups, and differences in the -OD-OS- values were calculated. Results: The whole sample showed a statistically significant inter-eye osmolarity difference (p=0.025; -OD-OS- = 9.2 ± 9.3 mOsm/l) and the correlation between Ocular Surface Disease Index and -OD-OS- (r=0.369; p<0.001). A statistically significant difference was found in the -OD-OS- value between symptomatic subgroups (Kruskal-Wallis, p=0.003). Mann-Whitney U test showed a significant difference between asymptomatic vs. moderate (p=0.006) vs. severe symp tomatic patients (p=0.001) and between mild vs. severe symptomatic patients (p=0.045), whereas no difference on -OD-OS- was found between participants with contiguous symptomatic subgroups (all p³0.174). Conclusion: Tear film inter-eye osmolarity differences are significantly higher in severe dry eye disease symptoms.

RESUMO Objetivo: Analisar se as diferenças entre osmolaridade entre os olhos foram relacionadas à sintomatologia do olho seco. Métodos: Um total de 135 participantes foram recrutados aleatoriamente entre os indivíduos da Clínica de Optometria da Faculdade de Optometria (Universidade de Santiago de Compostela). Em uma única sessão agendada após o recrutamento, o Índice de Doenças da Superfície Ocular foi preenchido seguindo as instruções padrão e as mensurações do TearLab foram feitas em ambos os olhos dos participantes (lapso de 10 a 15 min). Os valores de osmolaridade foram com parados entre os olhos direito e o esquerdo e a diferença absoluta ocular (-OD-OS-) correlacionada com a pontuação do Índice de Doença da Superfície Ocular para toda a amostra. Com base na pontuação do Índice de Doença da Superfície Ocular, a amostra foi dividida em quatro subgrupos sintomáticos, e as diferenças nos -OD-OS- os valores foram calcula dos. Resultados: A amostra total mostrou uma diferença de osmolarida de entre os olhos estatisticamente significativa (p=0,025; -OD-OS- = 9,2 ± 9,3 mOsm/l) e a correlação entre o Índice de Doença da Superfície Ocular e -OD-OS- (r=0,369; p<0,001). Diferença estatisticamente significativa foi encontrada no valor -OD-OS- entre os subgrupos sintomáticos (Kruskal-Wallis, p=0,003). O teste U de Mann-Whitney mostrou uma diferença significativa entre pacientes assintomáticos versus moderados (p=0,006) versus sintomáticos graves (p=0,001) e entre pacientes sinto máticos leves e graves (p=0,045), enquanto que nenhuma di ferença de -OD-OS- foi encontrada entre os participantes de subgrupos sintomáticos contíguos (todos p³0,174). Conclusão: As diferenças entre osmolaridade inter-ocular do filme lacrimal são significativamente maiores nos sintomas graves da doença do olho seco.

Oral bioaccessibility and human health risk assessment of trace elements in agricultural soils impacted by acid mine drainage.

Cultivated soils around the historic mine site of Tharsis (Spain) contain elevated concentrations of As (up to 621 mg kg-1), Cu (752 mg kg-1) and Pb (2395 mg kg-1), exceeding the regional background levels and the statutory limits set for agricultural use. A site-specific health risk assessment of occupational and environmental exposures was conducted using an approach based on guidelines from regulatory agencies, refined by combining bioaccessibility and bioavailability data. Oral bioaccessibility, as determined by simulating the human digestion process in vitro (Unified BARGE Method), was largely related to total trace element concentrations in soil. Arsenic seemed to be evenly distributed among the gastric and gastro-intestinal phases (about 31%), whereas the bioaccessible fraction of pH-dependent metal cations, like Pb and Zn, was noticeably higher in the stomach (nearly 50%) than in the gastro-intestinal tract (less than 10%). Bioaccessibility assessed by single extraction with 0.43 M HNO3 was overestimated by a factor of 1.2-1.4 relative to that obtained from the BARGE method. Site-specific relative bioavailability (RBA) values of As (27.7%) and Pb (42.6%), predicted from bioaccessibility measurements through linear regression models, had little effect on the overall risk estimates. For the ingestion pathway, the RBA-adjusted cancer risk values (9.7E-05 to 2.0E-04) exceeded the regulatory threshold in all plots, and the hazard index re-calculated after adjustment of oral dose was also above the allowable limit, with values ranging from 2.5 to 4.8. However, no detrimental health effects are expected to occur through inhalation of soil particles in nearby residents.

Phospho-RNA-seq: a modified small RNA-seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma.

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non-coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA-seq protocols due to lack of 5' phosphate or presence of 3' phosphate. These fragments were revealed using a modified protocol ("phospho-RNA-seq") incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5' and 3' ends, as well as human plasma exRNA Analyzing phospho-RNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow- and liver-enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-RNA-seq opens up new possibilities for plasma transcriptomic biomarker development.

Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

Droplet Digital PCR for Absolute Quantification of Extracellular MicroRNAs in Plasma and Serum: Quantification of the Cancer Biomarker hsa-miR-141.

Droplet-based digital PCR provides high-precision, absolute quantification of nucleic acid target sequences with wide-ranging applications for both research and clinical diagnostic applications. Droplet-based digital PCR enables absolute quantification by counting nucleic acid molecules encapsulated in discrete, volumetrically defined water-in-oil droplet partitions. The current available systems overcome the previous lack of scalable and practical technologies for digital PCR implementation. Extracellular microRNAs in biofluids (plasma, serum, urine, cerebrospinal fluid, etc.) are promising noninvasive biomarkers in multiple diseases and different clinical settings (e.g., diagnosis, early diagnosis, prediction of recurrence, and prognosis). Here we describe a protocol that enables highly precise and reproducible absolute quantification of extracellular microRNAs using droplet digital PCR.

Zoledronic Acid Treatment in Primary Bone Marrow Edema Syndrome.

Primary bone marrow edema syndrome (BMES) is characterized by the combination of joint pain and distinctive magnetic resonance imaging changes. It has been suggested that the use of bisphosphonate drugs reduce symptom severity. Our objective was to review cases of patients diagnosed with BMES in the last 7 years who had been treated with zoledronic acid. Access to a pharmaceutical database was gained in order to obtain a list of zoledronic acid prescriptions. Based on clinical and MRI criteria for BMES, patients were selected. Baseline pain intensity was evaluated on a scale of 0 to 3 and was also assessed after 3 and 12 months. Functional recovery was evaluated by noting if a patient had returned to carrying out his or her normal daily activities. Out of 633 patients, 17 cases of BMES were identified (8 men), with a median age of 54 ± 14.1 years. The most frequently affected joint was the ankle (9), followed by the hip. Sixteen patients presented with moderate to severe pain initially. Of those patients, 13 had no pain after 12 months. Zoledronic acid is a option in the management of BMES, since 75% of patients treated with it presented with a complete response.

Release of Cell-free MicroRNA Tumor Biomarkers into the Blood Circulation with Pulsed Focused Ultrasound: A Noninvasive, Anatomically Localized, Molecular Liquid Biopsy.

Purpose To compare the abilities of three pulsed focused ultrasound regimes (that cause tissue liquefaction, permeabilization, or mild heating) to release tumor-derived microRNA into the circulation in vivo and to evaluate release dynamics. Materials and Methods All rat experiments were approved by the University of Washington Institutional Animal Care and Use Committee. Reverse-transcription quantitative polymerase chain reaction array profiling was used to identify candidate microRNA biomarkers in a rat solid tumor cell line. Rats subcutaneously grafted with these cells were randomly assigned among three pulsed focused ultrasound treatment groups: (a) local tissue liquefaction via boiling histotripsy, (b) tissue permeabilization via inertial cavitation, and (c) mild (<10°C) heating of tissue, as well as a sham-treated control group. Blood specimens were drawn immediately prior to treatment and serially over 24 hours afterward. Plasma microRNA was quantified with reverse-transcription quantitative polymerase chain reaction, and statistical significance was determined with one-way analysis of variance (Kruskal-Wallis and Friedman tests), followed by the Dunn multiple-comparisons test. Results After tissue liquefaction and cavitation treatments (but not mild heating), plasma quantities of candidate biomarkers increased significantly (P value range, <.0001 to .04) relative to sham-treated controls. A threefold to 32-fold increase occurred within 15 minutes after initiation of pulsed focused ultrasound tumor treatment, and these increases persisted for 3 hours. Histologic examination confirmed complete liquefaction of the targeted tumor area with boiling histotripsy, in addition to areas of petechial hemorrhage and tissue disruption by means of cavitation-based treatment. Conclusion Mechanical tumor tissue disruption with pulsed focused ultrasound-induced bubble activity significantly increases the plasma abundance of tumor-derived microRNA rapidly after treatment. © RSNA, 2016 Online supplemental material is available for this article.

Ultra-Specific Isolation of Circulating Tumor Cells Enables Rare-Cell RNA Profiling.

The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra-pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.

Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation.

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.