scholarly journals Feasibility of phosphoproteomics on leftover samples after RNA extraction with guanidinium thiocyanate

2020 ◽  
Author(s):  
Frank Rolfs ◽  
Sander R. Piersma ◽  
Mariana Paes Dias ◽  
Jos Jonkers ◽  
Connie R. Jimenez
Keyword(s):  
Large Scale ◽  
Protein Extraction ◽  
Rna Extraction ◽  
Daily Practice ◽  
Extraction Methods ◽  
Small Samples ◽  
Data Types ◽  
Input Material ◽  
Guanidinium Thiocyanate ◽  
Class 1

AbstractIn daily practice, different types of biomolecules are usually extracted for large-scale ‘omics’ analysis with tailored protocols. However, when sample material is limited, an all-in-one strategy is preferable. While lysis of cells and tissues with urea is the accepted standard for phosphoproteomic applications, DNA, RNA and proteins can be simultaneously extracted from small samples using acid guanidinium thiocyanate-phenol-chloroform (AGPC). Use of AGPC for mass spectrometry (MS)-based phosphoproteomics has been reported, but not benchmarked. Here we compared urea-with AGPC-based protein extraction, profiling phosphorylations in the DNA damage response pathway after ionizing irradiation of U2OS cells as proof of principle. On average we identified circa 9000 phosphosites per sample with both extraction methods. Moreover, we observed high similarity of phosphosite characteristics (e.g. 94% shared class 1 identifications) and deduced kinase activities (e.g. ATM, ATR, CHEK1/2, PRKDC). AGPC-based sample extraction can thus replace standard cell lysates for phosphoproteomic workflows and may thus be an attractive way to obtain input material for multiple omics workflows, yielding several data types from a single sample.

scholarly journals Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yulia Newton ◽  
Andrew J. Sedgewick ◽  
Luis Cisneros ◽  
Justin Golovato ◽  
Mark Johnson ◽  
...  
Keyword(s):  
Large Scale ◽  
Clinical Decision Making ◽  
Rna Extraction ◽  
Transcriptome Profiling ◽  
Clinical Decision ◽  
Extraction Methods ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

Abstract Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

scholarly journals A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Catia Mio ◽  
Adriana Cifù ◽  
Stefania Marzinotto ◽  
Natascha Bergamin ◽  
Chiara Caldana ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Cost Effective ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Proteinase K ◽  
Rt Pcr ◽  
Isolation Method ◽  
Pcr Assay ◽  
Sensitivity Specificity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

scholarly journals Critical Aspects Concerning the Development of a Pooling Approach for SARS-CoV-2 Diagnosis Using Large-Scale PCR Testing

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 902
Author(s):  
Daniel Cruceriu ◽  
Oana Baldasici ◽  
Loredana Balacescu ◽  
Stefana Gligor-Popa ◽  
Mirela Flonta ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Diagnostic Assay ◽  
Assay Sensitivity ◽  
Infected People ◽  
Multiple Parameters ◽  
Accurate Procedure ◽  
Manual Methods ◽  
Pooling Strategy ◽  
Critical Aspects

The primary approach to controlling the spread of the pandemic SARS-CoV-2 is to diagnose and isolate the infected people quickly. Our paper aimed to investigate the efficiency and the reliability of a hierarchical pooling approach for large-scale PCR testing for SARS-CoV-2 diagnosis. To identify the best conditions for the pooling approach for SARS-CoV-2 diagnosis by RT-qPCR, we investigated four manual methods for both RNA extraction and PCR assessment targeting one or more of the RdRp, N, S, and ORF1a genes, by using two PCR devices and an automated flux for SARS-CoV-2 detection. We determined the most efficient and accurate diagnostic assay, taking into account multiple parameters. The optimal pool size calculation included the prevalence of SARS-CoV-2, the assay sensitivity of 95%, an assay specificity of 100%, and a range of pool sizes of 5 to 15 samples. Our investigation revealed that the most efficient and accurate procedure for detecting the SARS-CoV-2 has a detection limit of 2.5 copies/PCR reaction. This pooling approach proved to be efficient and accurate in detecting SARS-CoV-2 for all samples with individual quantification cycle (Cq) values lower than 35, accounting for more than 94% of all positive specimens. Our data could serve as a comprehensive practical guide for SARS-CoV-2 diagnostic centers planning to address such a pooling strategy.

scholarly journals Integrated Metabolomics and Transcriptomics Using an Optimised Dual Extraction Process to Study Human Brain Cancer Cells and Tissues

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 240
Author(s):  
Alison Woodward ◽  
Alina Pandele ◽  
Salah Abdelrazig ◽  
Catherine A. Ortori ◽  
Iqbal Khan ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Extraction Method ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Extraction Process ◽  
Extraction Methods ◽  
Serum Starvation ◽  
Extraction Techniques ◽  
Metabolic Genes ◽  
Dual Extraction

The integration of untargeted metabolomics and transcriptomics from the same population of cells or tissue enhances the confidence in the identified metabolic pathways and understanding of the enzyme–metabolite relationship. Here, we optimised a simultaneous extraction method of metabolites/lipids and RNA from ependymoma cells (BXD-1425). Relative to established RNA (mirVana kit) or metabolite (sequential solvent addition and shaking) single extraction methods, four dual-extraction techniques were evaluated and compared (methanol:water:chloroform ratios): cryomill/mirVana (1:1:2); cryomill-wash/Econospin (5:1:2); rotation/phenol-chloroform (9:10:1); Sequential/mirVana (1:1:3). All methods extracted the same metabolites, yet rotation/phenol-chloroform did not extract lipids. Cryomill/mirVana and sequential/mirVana recovered the highest amounts of RNA, at 70 and 68% of that recovered with mirVana kit alone. sequential/mirVana, involving RNA extraction from the interphase of our established sequential solvent addition and shaking metabolomics-lipidomics extraction method, was the most efficient approach overall. Sequential/mirVana was applied to study a) the biological effect caused by acute serum starvation in BXD-1425 cells and b) primary ependymoma tumour tissue. We found (a) 64 differentially abundant metabolites and 28 differentially expressed metabolic genes, discovering four gene-metabolite interactions, and (b) all metabolites and 62% lipids were above the limit of detection, and RNA yield was sufficient for transcriptomics, in just 10 mg of tissue.

scholarly journals Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2625
Author(s):  
Muzammeer Mansor ◽  
Jameel R. Al-Obaidi ◽  
Nurain Nadiah Jaafar ◽  
Intan Hakimah Ismail ◽  
Atiqah Farah Zakaria ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Chloroform Solution ◽  
Milk Proteins ◽  
Peptide Mass Fingerprinting ◽  
Extraction Methods ◽  
Dairy Goats ◽  
Goat’S Milk ◽  
Peptide Mass ◽  
Goat's Milk

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

scholarly journals Comparison of RNA Extraction Methods for Molecular Analysis of Oral Cytology

2016 ◽  
Vol 50 (2) ◽  
pp. 108-115 ◽  
Author(s):  
Alves Mônica Ghislaine Oliveira ◽  
Mario Pérez-Sayáns ◽  
Maria-Elena Padín-Iruegas ◽  
Maria Dolores Reboiras-López ◽  
José Manuel Suarez-Peñaranda ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Extraction Methods ◽  
Oral Cytology

scholarly journals W.S. Gosset and Some Neglected Concepts in Experimental Statistics: Guinnessometrics II

2011 ◽  
Vol 6 (2) ◽  
pp. 252-277 ◽  
Author(s):  
Stephen T. Ziliak
Keyword(s):  
Experimental Design ◽  
Sample Size ◽  
Large Scale ◽  
Statistical Significance ◽  
Small Sample ◽  
Small Samples ◽  
Significant Advance ◽  
Economic Approach ◽  
Barley Malt ◽  
Level Of Significance

AbstractStudent's exacting theory of errors, both random and real, marked a significant advance over ambiguous reports of plant life and fermentation asserted by chemists from Priestley and Lavoisier down to Pasteur and Johannsen, working at the Carlsberg Laboratory. One reason seems to be that William Sealy Gosset (1876–1937) aka “Student” – he of Student'st-table and test of statistical significance – rejected artificial rules about sample size, experimental design, and the level of significance, and took instead an economic approach to the logic of decisions made under uncertainty. In his job as Apprentice Brewer, Head Experimental Brewer, and finally Head Brewer of Guinness, Student produced small samples of experimental barley, malt, and hops, seeking guidance for industrial quality control and maximum expected profit at the large scale brewery. In the process Student invented or inspired half of modern statistics. This article draws on original archival evidence, shedding light on several core yet neglected aspects of Student's methods, that is, Guinnessometrics, not discussed by Ronald A. Fisher (1890–1962). The focus is on Student's small sample, economic approach to real error minimization, particularly in field and laboratory experiments he conducted on barley and malt, 1904 to 1937. Balanced designs of experiments, he found, are more efficient than random and have higher power to detect large and real treatment differences in a series of repeated and independent experiments. Student's world-class achievement poses a challenge to every science. Should statistical methods – such as the choice of sample size, experimental design, and level of significance – follow the purpose of the experiment, rather than the other way around? (JEL classification codes: C10, C90, C93, L66)

Comparison of Two Protein Extraction Methods for Tea Residues

2011 ◽  
Vol 138-139 ◽  
pp. 933-936 ◽  
Author(s):  
Xuan Chen ◽  
Hong Yu Luo ◽  
Jun Yu ◽  
Peng Xiang Yue ◽  
Lin Zhou ◽  
...  
Keyword(s):  
High Temperature ◽  
Protein Extraction ◽  
Ethanol Concentration ◽  
Extraction Methods ◽  
Extraction Yield ◽  
Factorial Experiments ◽  
Extraction Technology ◽  
Digestion Method ◽  
Solid Ratio ◽  
Lower Temperature

Alcohol-alkali method and base digestion method were investigated to extract proteins from tea residues, respectively. According to single factorial experiments, results showed that the optimal extraction technology of alcohol-alkali method were pH 12, temperature of 80 °C, ethanol concentration of 60%, liquid-solid ratio of 40, 60 min, and the protein extraction rate reached 15.0%. And the optimal extract conditions of base digestion were pH 12, temperature of 80 °C, liquid-solid ratio of 50, 80 min, which made the protein yield reached 31.5%. Furthermore, alcohol-alkali method was more beneficial to protein extraction from tea residues under lower temperature and weak alkali condition (40-60 °C, pH 8-10). While base digestion had higher extraction yield under high temperature and strong alkali condition (60-80 °C, pH 11-12).

scholarly journals Exploring the feasibility of a network of organizations for pain rehabilitation: what are the lessons learned?

2020 ◽  
Author(s):  
Cynthia Lamper ◽  
Ivan PJ Huijnen ◽  
Mariëlle EAL Kroese ◽  
Albère J Köke ◽  
Gijs Brouwer ◽  
...  
Keyword(s):  
Health Care ◽  
Health Care Professionals ◽  
Large Scale ◽  
Critical Mass ◽  
Implementation Strategies ◽  
Daily Practice ◽  
Lessons Learned ◽  
Integration Of Care ◽  
Treatment Protocols ◽  
Rehabilitation Care

Abstract Background and aims: Integration of care is lacking for chronic musculoskeletal pain (CMP) patients. Network Pain Rehabilitation Limburg (NPRL), a transmural health care network, has been designed to provide integrated rehabilitation care from a biopsychosocial perspective to improve patients’ levels of functioning. This feasibility study aims to provide insight into barriers and facilitators for the development, implementation, and transferability of NPRL.Methods: This study was conducted with a three-phase iterative and incremental design from October 2017 to October 2018. NPRL comprises two rehabilitation practices, and three local primary care networks, with a general practitioner together with, a mental health practice nurse, and a physiotherapist or exercise therapist. These stakeholders with a random sample of participating patients took part in evaluations, consisting of interviews, focus groups, and observations. Field notes and observations were recorded during meetings. The Consolidated Framework for Implementation Research guided data collection and analysis. Results were used to refine the next phase.Results: According to health care professionals (HCPs), guidelines and treatment protocols facilitate consistency and transparency in collaboration, biopsychosocial language, and treatment. One barrier is stigmatization of CMP in society. Non-participating HCPs’ treatment approaches are often more biomedical than biopsychosocial, causing patients to resist participating in NPRL. The current organization of health care, with cultural, structural, and financial aspects, acts as a barrier, complicating implementation between and within practices. HCPs preferred the iterative, bottom-up strategy. A critical mass of participating organizations is needed for proper implementation.Conclusion: NPRL is feasible in daily practice if barriers are overcome and facilitators of development, implementation, and transferability are promoted. These findings will be used to refine NPRL. A large-scale process and effect evaluation will be performed. Our implementation strategies and results may assist other health care organizations aspiring to implement a transmural network using a similar model.

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