scholarly journals Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Kevin Kuhlmann ◽  
Melanie Cieselski ◽  
Julia Schumann
Keyword(s):  
Dynamic Range ◽  
Genetic Material ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Test Procedures ◽  
Experimental Conditions ◽  
Rna Quantification ◽  
The Stability ◽  
Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

scholarly journals Clarity Plus™ digital PCR: A novel platform for absolute quantification of SARS-CoV-2

2021 ◽  
Author(s):  
Shawn Yi Han Tan ◽  
Milton Sheng Yi Kwek ◽  
Huiyu Low ◽  
Yan Ling Joy Pang
Keyword(s):  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Viral Loads ◽  
Nucleic Acid Analysis ◽  
Polymerase Chain ◽  
The Absolute ◽  
Assay Precision ◽  
Digital Polymerase Chain Reaction ◽  
Higher Sensitivity

In recent years, the usage of digital polymerase chain reaction (dPCR) for various clinical applications has increased exponentially. Considering the growing demand for improved dPCR technology, the Clarity Plus™ dPCR system which features enhanced multiplexing capability and a wider dynamic range for nucleic acid analysis was recently launched. In this study, a dPCR assay optimized for use on Clarity Plus™ was evaluated for the absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent responsible for the global coronavirus disease 2019 (COVID-19) outbreak. The assay demonstrated good inter- and intra- assay precision, accuracy, as well as excellent linearity across a range of over 6 orders of magnitude for target gene quantification. In addition, comparison of the assay on both dPCR and qPCR platforms revealed that dPCR exhibited a slightly higher sensitivity compared to its qPCR counterpart when quantifying SARS-CoV-2 at a lower concentration. Overall, the results showed that the dPCR assay is a reliable and effective approach for the absolute quantification of SARS-CoV-2 and can potentially be adopted as a molecular tool in applications such as detecting low viral loads in patients as well as in wastewater surveillance of COVID-19.

scholarly journals A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR

Micromachines ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1025
Author(s):  
Gangwei Xu ◽  
Huaqing Si ◽  
Fengxiang Jing ◽  
Peng Sun ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Microfluidic Chip ◽  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Mutual Interference ◽  
Fluorescence Signal ◽  
Pdms Layer ◽  
Glass Substrates ◽  
Planar Area ◽  
Dna Template

In this work, a double-deck microfluidic chip was presented for digital PCR application. This chip consists of two reverse-placed micro-patterned polydimethylsiloxane (PDMS) layers between the top and bottom glass substrates. Each micropatterned PDMS layer contains more than 20,000 cylindrical micro-chambers to hold the partitioned droplets. The double-deck designs can double the number of chambers and reagent capacity without changing the planar area of the chip. In addition, carbon black was mixed into the pure PDMS gel to obstruct the passage of fluorescence from the positive chambers between the two layers of the chip. Thus, the fluorescence signal of micro-chambers in different layers of the chip after PCR can be collected without mutual interference. The quantitative capability of the proposed chip was evaluated by measuring a 10-fold serial dilution of the DNA template. A high accuracy of the absolute quantification for nucleic acid with a dynamic range of 105 was demonstrated by this chip in this work. Owing to its characteristics of small planar area, large capacity, and sensitivity, the double-deck microfluidic chip is expected to further promote the extensive applications of digital PCR.

scholarly journals A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

2020 ◽  
Author(s):  
Xiaolin Wu ◽  
Cheryl Chan ◽  
Yie Hou Lee ◽  
Stacy L. Springs ◽  
Timothy K. Lu ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Virus Detection ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Cross Reactivity ◽  
Widespread Application ◽  
Virus Particles ◽  
Barr Virus ◽  
The Absolute ◽  
Epstein Barr

AbstractQuantitative real-time PCR and CRISPR-based methods detect SARS-CoV-2 in 1 hour but do not allow for the absolute quantification of virus particles, which could reduce inter-lab variability and accelerate research. The 4-hour reaction time of the existing digital PCR-based method for absolute virus quantification is too long for widespread application. We report a RApid DIgital Crispr Approach (RADICA) for the absolute quantification of SARS-CoV-2 DNA and Epstein–Barr virus DNA in human samples that yields results within 1 hour. For validation, we compared RADICA to digital PCR for quantifying synthetic SARS-CoV-2 DNA and Epstein–Barr viral DNA. RADICA allows absolute quantification of DNA with a dynamic range from 0.6 to 2027 copies/µL (R2 value > 0.98), without cross-reactivity on similar virus or human background DNA. Thus, RADICA can accurately detect and quantify nucleic acid in 1h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based virus detection.

scholarly journals Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes

2021 ◽  
Author(s):  
Joan Xiaohui Yang ◽  
Xin Yuan Zhao ◽  
Dexi Bi ◽  
Citra Mattar ◽  
Joy Yan Ling Pang ◽  
...  
Keyword(s):  
New Technologies ◽  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Variable Expression ◽  
Sample Extraction ◽  
Relative Standard ◽  
Extracellular Mirna ◽  
Digital Polymerase Chain Reaction

Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 – 33 copies/µL (r2=0.999), high accuracy and excellent reproducibility of <10% relative standard deviation. The method then identified the variable expression of Gapdh (0.72–16.95 copies/oocyte), Hprt1 (1.05–19.05 copies/oocyte) and ATPase 6, (5.55–32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

scholarly journals A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach

2021 ◽  
Vol 22 (3) ◽  
pp. 1017
Author(s):  
Zhongjie Fei ◽  
Rongbin Wei ◽  
Chu Cheng ◽  
Pengfeng Xiao
Keyword(s):  
Real Time ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Global Public Health ◽  
Reaction Volume ◽  
Novel Approach ◽  
Highly Sensitive ◽  
Target Rna

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.

Use of a Lyophilized Reference Plasma to Compare Coagulation Test Procedures

1975 ◽  
Vol 34 (02) ◽  
pp. 426-444 ◽  
Author(s):  
J Kahan ◽  
I Nohén
Keyword(s):  
Oral Anticoagulant Therapy ◽  
Repeated Measurements ◽  
Test Procedures ◽  
Coagulation Activity ◽  
Close Relationship ◽  
Measured Activity ◽  
Test Materials ◽  
The Difference ◽  
The Stability ◽  
The One

SummaryIn 4 collaborative trials, involving a varying number of hospital laboratories in the Stockholm area, the coagulation activity of different test materials was estimated with the one-stage prothrombin tests routinely used in the laboratories, viz. Normotest, Simplastin-A and Thrombotest. The test materials included different batches of a lyophilized reference plasma, deep-frozen specimens of diluted and undiluted normal plasmas, and fresh and deep-frozen specimens from patients on long-term oral anticoagulant therapy.Although a close relationship was found between different methods, Simplastin-A gave consistently lower values than Normotest, the difference being proportional to the estimated activity. The discrepancy was of about the same magnitude on all the test materials, and was probably due to a divergence between the manufacturers’ procedures used to set “normal percentage activity”, as well as to a varying ratio of measured activity to plasma concentration. The extent of discrepancy may vary with the batch-to-batch variation of thromboplastin reagents.The close agreement between results obtained on different test materials suggests that the investigated reference plasma could be used to calibrate the examined thromboplastin reagents, and to compare the degree of hypocoagulability estimated by the examined PIVKA-insensitive thromboplastin reagents.The assigned coagulation activity of different batches of the reference plasma agreed closely with experimentally obtained values. The stability of supplied batches was satisfactory as judged from the reproducibility of repeated measurements. The variability of test procedures was approximately the same on different test materials.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals Low-Energy Electron Damage to Condensed-Phase DNA and Its Constituents

2021 ◽  
Vol 22 (15) ◽  
pp. 7879
Author(s):  
Yingxia Gao ◽  
Yi Zheng ◽  
Léon Sanche
Keyword(s):  
Condensed Phase ◽  
Genetic Material ◽  
High Energy ◽  
Dna Lesions ◽  
Low Energy ◽  
Experimental Investigations ◽  
Experimental Conditions ◽  
Strand Breaks ◽  
Sequence Of Events ◽  
Wide Range

The complex physical and chemical reactions between the large number of low-energy (0–30 eV) electrons (LEEs) released by high energy radiation interacting with genetic material can lead to the formation of various DNA lesions such as crosslinks, single strand breaks, base modifications, and cleavage, as well as double strand breaks and other cluster damages. When crosslinks and cluster damages cannot be repaired by the cell, they can cause genetic loss of information, mutations, apoptosis, and promote genomic instability. Through the efforts of many research groups in the past two decades, the study of the interaction between LEEs and DNA under different experimental conditions has unveiled some of the main mechanisms responsible for these damages. In the present review, we focus on experimental investigations in the condensed phase that range from fundamental DNA constituents to oligonucleotides, synthetic duplex DNA, and bacterial (i.e., plasmid) DNA. These targets were irradiated either with LEEs from a monoenergetic-electron or photoelectron source, as sub-monolayer, monolayer, or multilayer films and within clusters or water solutions. Each type of experiment is briefly described, and the observed DNA damages are reported, along with the proposed mechanisms. Defining the role of LEEs within the sequence of events leading to radiobiological lesions contributes to our understanding of the action of radiation on living organisms, over a wide range of initial radiation energies. Applications of the interaction of LEEs with DNA to radiotherapy are briefly summarized.

scholarly journals Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kim Hoa Ho ◽  
Annarita Patrizi
Keyword(s):  
Gene Expression ◽  
Choroid Plexus ◽  
Sensory Deprivation ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Experimental Conditions ◽  
Brain Repair ◽  
Expression Studies ◽  
Testing Algorithms ◽  
Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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