Journal of biological methods最新文献

{"title":"Comparing NGS and NanoString platforms in peripheral blood mononuclear cell transcriptome profiling for advanced heart failure biomarker development.","authors":"Galyna Bondar, Wenjie Xu, David Elashoff, Xinmin Li, Emmanuelle Faure-Kumar, Tra-Mi Bao, Tristan Grogan, Jim Moose, Mario C Deng","doi":"10.14440/jbm.2020.300","DOIUrl":"10.14440/jbm.2020.300","url":null,"abstract":"<p><p>In preparation to create a clinical assay that predicts 1-year survival status of advanced heart failure (AdHF) patients before surgical/interventional therapies and to select the appropriate clinical assay platform for the future assay, we compared the properties of next generation sequencing (NGS) used in the gene discovery phase to the NanoString platform used in the clinical assay development phase. In 25 AdHF patients in a tertiary academic medical center from 2015 to 2016, PBMC samples were collected and aliquoted for NGS RNA whole transcriptome sequencing and compared to 770 genes represented on NanoString's PanCancer IO 360 Gene Expression research panel. Prior to statistical analysis, NanoString and NGS expression values were log transformed. We computed Pearson correlation coefficients for each sample, comparing gene expression values between NanoString and NGS across the set of matched genes and for each of the matched genes across the set of samples. Genes were grouped by average NGS expression, and the NanoString-NGS correlation for each group was computed. Out of 770 genes from the NanoString panel, 734 overlapped between both platforms and showed high intrasample correlation. Within an individual sample, there was an expression-level dependent correlation between both platforms. The low- <i>vs</i>. intermediate/high-expression groups showed NGS average correlation 0.21 <i>vs</i>. 0.58-0.68, respectively, and NanoString average correlation 0.07-0.34 <i>vs</i>. 0.59-0.70, respectively. NanoString demonstrated high reproducibility (<i>R</i> ² > 0.99 for 100 ng input), sensitivity (probe counts between 100 and 500 detected and quantified), and robustness (similar gene signature scores across different RNA input concentrations, cartridges, and outcomes). Data from NGS and NanoString were highly correlated. These platforms play a meaningful, complementary role in the biomarker development process.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 1","pages":"e123"},"PeriodicalIF":0.0,"publicationDate":"2020-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/fa/d4/jbm-7-1-e123.PMC6974694.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37575405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A simplified design for the <i>C. elegans</i> lifespan machine.","authors":"Mark Abbott,&nbsp;Stephen A Banse,&nbsp;Ilija Melentijevic,&nbsp;Cody M Jarrett,&nbsp;Jonathan St Ange,&nbsp;Christine A Sedore,&nbsp;Ron Falkowski,&nbsp;Benjamin W Blue,&nbsp;Anna L Coleman-Hulbert,&nbsp;Erik Johnson,&nbsp;Max Guo,&nbsp;Gordon J Lithgow,&nbsp;Patrick C Phillips,&nbsp;Monica Driscoll","doi":"10.14440/jbm.2020.332","DOIUrl":"https://doi.org/10.14440/jbm.2020.332","url":null,"abstract":"<p><p><i>Caenorhabditis elegans</i> (<i>C. elegans</i>) lifespan assays constitute a broadly used approach for investigating the fundamental biology of longevity. Traditional <i>C. elegans</i> lifespan assays require labor-intensive microscopic monitoring of individual animals to evaluate life/death over a period of weeks, making large-scale high throughput studies impractical. The lifespan machine developed by Stroustrup <i>et al</i>. (2013) adapted flatbed scanner technologies to contribute a major technical advance in the efficiency of <i>C. elegans</i> survival assays. Introducing a platform in which large portions of a lifespan assay are automated enabled longevity studies of a scope not possible with previous exclusively manual assays and facilitated novel discovery. Still, as initially described, constructing and operating scanner-based lifespan machines requires considerable effort and expertise. Here we report on design modifications that simplify construction, decrease cost, eliminate certain mechanical failures, and decrease assay workload requirements. The modifications we document should make the lifespan machine more accessible to interested laboratories.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"7 4","pages":"e137"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/fc/15/jbm-7-4-e137.PMC7666331.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9153236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum: A descriptive guide for absolute quantification of produced shRNA pseudotyped lentiviral particles by real-time PCR.","authors":"Virginie Mournetas,&nbsp;Sofia Melo Pereira,&nbsp;David G Fernig,&nbsp;Patricia Murray","doi":"10.14440/jbm.2019.326","DOIUrl":"https://doi.org/10.14440/jbm.2019.326","url":null,"abstract":"<p><p>[This corrects the article on p. e55 in vol. 3, PMID: 31453218.].</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"6 4","pages":"e122"},"PeriodicalIF":0.0,"publicationDate":"2019-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/67/2b/jbm-6-4-e122.PMC6974693.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37575407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A multivariate statistical analysis of the effects of styrene maleic acid encapsulated RL71 in a xenograft model of triple negative breast cancer.","authors":"Orleans N K Martey,&nbsp;Khaled Greish,&nbsp;Paul F Smith,&nbsp;Rhonda J Rosengren","doi":"10.14440/jbm.2019.306","DOIUrl":"https://doi.org/10.14440/jbm.2019.306","url":null,"abstract":"<p><p>We have previously shown that the curcumin derivative 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidine-4-one (RL71), when encapsulated in styrene maleic acid micelles (SMA-RL71), significantly suppressed the growth of MDA-MB-231 xenografts by 67%. Univariate statistical analysis showed that pEGFR/EGFR, pAkt/Akt, pmTOR/mTOR and p4EBP1/4EPBP1 were all significantly decreased in tumors from treated mice compared to SMA controls. In this study, multivariate statistical analyses (MVAs) were performed to identify the molecular networks that worked together to drive tumor suppression, with the aim to determine if this analysis could also be used to predict treatment outcome. Linear discriminant analysis correctly predicted, to 100% certainty, mice that received SMA-RL71 treatment. Additionally, results from multiple linear regression showed that the expression of Ki67, PKC-α, PP2AA-α, PP2AA-β and CaD1 networked together to drive tumor growth suppression. Overall, the MVAs provided evidence for a molecular network of signaling proteins that drives tumor suppression in response to SMA-RL71 treatment, which should be explored further in animal studies of cancer.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"6 4","pages":"e121"},"PeriodicalIF":0.0,"publicationDate":"2019-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/29/83/jbm-6-4-e121.PMC6974696.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37575404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A standardized methodology for longitudinal assessment of corneal endothelial morphometry in eye banked corneas.","authors":"Zala Lužnik,&nbsp;Zhongmou Sun,&nbsp;Jia Yin,&nbsp;Beth Ann Benetz,&nbsp;Jonathan H Lass,&nbsp;Reza Dana","doi":"10.14440/jbm.2019.304","DOIUrl":"https://doi.org/10.14440/jbm.2019.304","url":null,"abstract":"<p><p>Eye banked research-grade human donor corneas serve as principal <i>ex vivo</i> source for studying the mechanisms that underlie corneal endothelial cell damage/death and survival. Wide-field specular microscopy can be used for corneal endothelial visualization and allows for indirect assessment of endothelial cell function by analyzing endothelial cell density and morphometric parameters. However, a standardized approach is needed to observe corneal endothelial changes over time. This protocol describes reliable <i>ex vivo</i> methods for consecutive analyses of human donor corneal endothelial cell density and morphometric parameters change using a wide-field dual imaging specular microscope. This protocol involves tissue warming, acquisition and analysis of specular endothelial images, assessment of corneal layers with the new Enhance mode, optical pachymetry measurement, and qualitative image quality grading scales. This quantitative and qualitative evaluation of donor corneas allows for a systematic analysis of endothelial dynamic responses to <i>ex vivo</i> induced stress and can be used as a valuable tool to better elucidate specular findings and mechanisms mediating corneal endothelial cell loss in corneal disease and after transplantation.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"6 4","pages":"e120"},"PeriodicalIF":0.0,"publicationDate":"2019-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/49/aa/jbm-6-4-e120.PMC6888603.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37502318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Embryo microinjection of the lecithotrophic sea urchin Heliocidaris erythrogramma","authors":"A. Edgar, M. Byrne, G. Wray","doi":"10.14440/jbm.2019.292","DOIUrl":"https://doi.org/10.14440/jbm.2019.292","url":null,"abstract":"Microinjection is a common embryological technique used for many types of experiments, including lineage tracing, manipulating gene expression, or genome editing. Injectable reagents include mRNA overexpression, mis-expression, or dominant-negative experiments to examine a gene of interest, a morpholino antisense oligo to prevent translation of an mRNA or spliceoform of interest and CRISPR-Cas9 reagents. Thus, the technique is broadly useful for basic embryological studies, constructing gene regulatory networks, and directly testing hypotheses about cis-regulatory and coding sequence changes underlying the evolution of development. However, the methods for microinjection in typical planktotrophic marine invertebrates may not work well in the highly modified eggs and embryos of lecithotrophic species. This protocol is optimized for the lecithotrophic sea urchin Heliocidaris erythrogramma.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49515011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"FairSubset: A tool to choose representative subsets of data for use with replicates or groups of different sample sizes","authors":"K. Ortell, Pawel M. Switonski, J. Delaney","doi":"10.14440/jbm.2019.299","DOIUrl":"https://doi.org/10.14440/jbm.2019.299","url":null,"abstract":"High-impact journals are promoting transparency of data. Modern scientific methods can be automated and produce disparate samples sizes. In many cases, it is desirable to retain identical or pre-defined sample sizes between replicates or groups. However, choosing which subset of originally acquired data that best matches the entirety of the data set without introducing bias is not trivial. Here, we released a free online tool, FairSubset, and its constituent Shiny App R code to subset data in an unbiased fashion. Subsets were set at the same N across samples and retained representative average and standard deviation information. The method can be used for quantitation of entire fields of view or other replicates without biasing the data pool toward large N samples. We showed examples of the tool’s use with fluorescence data and DNA-damage related Comet tail quantitation. This FairSubset tool and the method to retain distribution information at the single-datum level may be considered for standardized use in fair publishing practices.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41842911","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform","authors":"V. Iannarone, G. Cruz, B. Hilliard, M. Barbe","doi":"10.14440/jbm.2019.289","DOIUrl":"https://doi.org/10.14440/jbm.2019.289","url":null,"abstract":"Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure. In order to assess collagen synthesis and extracellular accumulation in fibrotic disorders, improved methods are needed to detect changes in procollagen versus mature collagen at the protein level. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. bis-Tris, gradient vs. non-gradient, sodium dodecyl sulfate (SDS) vs. no SDS); (2) sample preparation (SDS vs. no SDS, β-mercaptoethanol (BME) vs. no BME, boiling vs. no boiling); and (3) running buffer composition (SDS vs. no SDS). Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. The best resolution of type 1 procollagens is achieved using 4%–12% Tris-glycine gels without the presence of SDS in the gel itself, although SDS in the running and sample buffers are needed. Also, BME must not be added to the sample buffer and samples should not be boiled. For characterization of mature collagen 1(I), both 8% and gradients type gels are appropriate, although still without SDS, yet with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled. Boiling is to be avoided as the antigenic site recognized by the monoclonal antibody used is sensitive to thermal denaturation, as is the case with many monoclonal antibodies available on the market. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48173877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vivo measurement of enhanced agouti-related peptide release in the paraventricular nucleus of the hypothalamus through Gs activation of agouti-related peptide neurons","authors":"Z. Cui, Adam S. Smith","doi":"10.14440/jbm.2019.288","DOIUrl":"https://doi.org/10.14440/jbm.2019.288","url":null,"abstract":"Agouti-related peptide (AgRP) neurons of the hypothalamus play a role in hunger-triggered food intake, stability of body weight, and long-term energy balance. A recent study showed that activation of the Gs-linked G protein-coupled receptors (GCPR) expressed by hypothalamic AgRP neurons promotes a sustained increase in food intake. Enhanced AgRP release has been the postulated underlying mechanism. Here, we confirmed that activation of Gs-coupled receptors expressed by AgRP neurons in the arcuate nucleus (ARC) of the hypothalamus, which is the primary brain region for the synthesis and release of AgRP, leads to increased release of AgRP in the paraventricular nucleus of the hypothalamus (PVN). We were unable to confirm changes in AgRP expression or intracellular content using traditional histological techniques. Thus, we developed an assay to measure AgRP in the extracellular fluid in the brain using large molecular weight cut-off microdialysis probes. Our technique enables assessment of brain AgRP pharmacokinetics under physiological conditions and in response to specific pharmacological interventions designed to modulate AgRP signaling.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44014390","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative analysis of diagnostic platforms for measurement of differentially methylated insulin DNA","authors":"R. Farr, Wilson K. M. Wong, Cody-Lee Maynard, S. Tersey, R. Mirmira, A. Hardikar, M. Joglekar","doi":"10.14440/jbm.2019.280","DOIUrl":"https://doi.org/10.14440/jbm.2019.280","url":null,"abstract":"Circulating cell-free DNA (cfDNA) has been intensively investigated as a diagnostic and prognostic marker for various cancers. In recent years, presence of unmethylated insulin cfDNA in the circulation has been correlated with pancreatic β-cell death and risk of developing type 1 diabetes. Digital (d)PCR is an increasingly popular method of quantifying insulin cfDNA due to its ability to determine absolute copy numbers, and its increased sensitivity when compared to the more routinely used quantitative PCR. Multiple platforms have been developed to carry out dPCR. However, not all technologies perform comparably, thereby necessitating evaluation of each platform. Here, we compare two dPCR platforms: the QuantStudio 3D (QS3D, Applied Biosystems) and the QX200 (Bio-Rad), to measure copies of unmethylated/methylated insulin plasmids. The QS3D detected greater copy numbers of the plasmids than the QX200 (manual mode), whereas QX200 demonstrated minimal replicate variability, increased throughput, ease of use and the potential for automation. Overall, the performance of QX200, in our hands, was better suited to measure differentially methylated insulin cfDNA.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45386342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}