High-Throughput Single-Cell Real-Time Quantitative PCR Analysis

2019 ◽  
pp. 177-183
Author(s):  
Liora Haim-Vilmovsky
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Quantitative Pcr ◽  
High Throughput ◽  
Pcr Analysis ◽  
Real Time Quantitative Pcr

Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda

2017 ◽  
Vol 27 (1) ◽  
pp. 112-121 ◽  
Author(s):  
Zhongyang Sun ◽  
Jia Deng ◽  
Haizhen Wu ◽  
Qiyao Wang ◽  
Yuanxing Zhang
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Edwardsiella Tarda ◽  
Pcr Analysis ◽  
Real Time Quantitative Pcr ◽  
Selection Of

Human disease characterization: real-time quantitative PCR analysis of gene expression

2001 ◽  
Vol 6 (20) ◽  
pp. 1062-1067 ◽  
Author(s):  
James V Snider ◽  
Mark A Wechser ◽  
Izidore S Lossos
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Human Disease ◽  
Pcr Analysis ◽  
Real Time Quantitative Pcr

scholarly journals Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Yeast ◽  
2000 ◽  
Vol 1 (3) ◽  
pp. 201-210 ◽  
Author(s):  
A. Al-Taher ◽  
A. Bashein ◽  
T. Nolan ◽  
M. Hollingsworth ◽  
Brady G.
Keyword(s):  
Potassium Channel ◽  
Single Cell ◽  
Real Time ◽  
Quantitative Pcr ◽  
Single Cells ◽  
Initial Step ◽  
Small Samples ◽  
Rt Pcr ◽  
Real Time Quantitative Pcr ◽  
Global Amplification

We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use ofTaqMan™real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst108total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

scholarly journals Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos

2011 ◽  
Vol 23 (2) ◽  
pp. 353 ◽  
Author(s):  
Damien B. B. P. Paris ◽  
Ewart W. Kuijk ◽  
Bernard A. J. Roelen ◽  
Tom A. E. Stout
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Embryo Development ◽  
Reference Genes ◽  
Stable Expression ◽  
Pcr Analysis ◽  
Blastocyst Development ◽  
Real Time Quantitative Pcr

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.

scholarly journals Real-Time quantitative PCR analysis of factor XI mRNA variants in human platelets

2004 ◽  
Vol 2 (10) ◽  
pp. 1713-1719 ◽  
Author(s):  
A. Podmore ◽  
M. Smith ◽  
G. Savidge ◽  
A. Alhaq
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Human Platelets ◽  
Pcr Analysis ◽  
Factor Xi ◽  
Real Time Quantitative Pcr ◽  
Mrna Variants

scholarly journals Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

2014 ◽  
Vol 80 (17) ◽  
pp. 5439-5446 ◽  
Author(s):  
Wesley Loftie-Eaton ◽  
Allison Tucker ◽  
Ann Norton ◽  
Eva M. Top
Keyword(s):  
Flow Cytometry ◽  
Real Time ◽  
Quantitative Pcr ◽  
High Throughput ◽  
Performance Criteria ◽  
Plate Count ◽  
Real Time Quantitative Pcr ◽  
Advantages And Disadvantages ◽  
Free Cells ◽  
Loss Rates

ABSTRACTThe maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in threePseudomonashosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses.

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