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Gene Quantification Newsletter |
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Dear researcher,
dear Gene Quantification page reader,
Our newsletter informs about the latest news in quantitative real-time
PCR (qPCR and RT-qPCR), which are compiled and summarised on the Gene Quantification
homepage. The focus of this newsletter issue is:
- digital-PCR application papers - digital-PCR.gene-quantification.info
- real-time PCR optimisation - optimisation.gene-quantification.info
- GenEx version 5 - download a free trial version - GenEx.gene-quantification.info
Digital PCR (dPCR) is a refinement of conventional PCR methods that can be used to directly quantify and clonally amplify nucleic acids (including DNA, cDNA, methylated DNA, or RNA). The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences - such as copy number variants, point mutations, and it is routinely used for clonal amplification of samples for "next-generation sequencing."
... ... more about digital
here => digital-PCR.gene-quantification.info
application papers
The
Significance of Digital Gene Expression Profiles.
Stephane Audic &Jean-Michel Claverie, GENOMER ESEARCH (1997)
7: 986–995
Evaluation
of a droplet digital polymerase chain reaction format for DNA copy
number quantification.
Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S,
Emslie KR., Anal Chem. 2012 84(2): 1003-1011
Further
Improvement in Quantifying Male Fetal DNA in Maternal Plasma.
Jin S, Michelle Lin X, Law H, Kwek KY, Yeo GS, Ding C., Clin Chem. 2012
High-throughput
droplet digital PCR system for absolute quantitation of DNA copy number.
Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz
AJ, Bright IJ, Lucero MY, Hiddessen AL, et al., Anal Chem. 2011
Nov 15;83(22): 8604-8610
Multiplexed
quantification of nucleic acids with large dynamic range using
multivolume digital RT-PCR on a rotational SlipChip tested with HIV and
hepatitis C viral load.
Shen F, Sun B, Kreutz JE, Davydova EK, Du W, Reddy PL, Joseph LJ,
Ismagilov RF., J Am Chem Soc. 2011 Nov 9;133(44): 17705-17712
Theoretical
design and analysis of multivolume digital assays with wide dynamic
range validated experimentally with microfluidic digital PCR.
Kreutz JE, Munson T, Huynh T, Shen F, Du W, Ismagilov RF., Anal Chem.
2011 Nov 1;83(21): 8158-8168
Digital
PCR provides absolute quantitation of viral load for an occult RNA
virus.
White RA 3rd, Quake SR, Curr K., J Virol Methods. 2012 179(1): 45-50
1-Million
droplet array with wide-field fluorescence imaging for digital PCR.
Hatch AC, Fisher JS, Tovar AR, Hsieh AT, Lin R, Pentoney SL, Yang DL,
Lee AP., Lab Chip. 2011 11(22): 3838-3845
Implementing
prenatal diagnosis based on cell-free fetal DNA: accurate
identification of factors affecting fetal DNA yield.
Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS., PLoS One.
2011;6(10): e25202
Megapixel digital PCR.
Heyries KA, Tropini C, Vaninsberghe M, Doolin C, Petriv OI, Singhal A,
Leung K, Hughesman CB, Hansen CL., Nat Methods. 2011 8(8): 649-651
... ... more about digital here => digital-PCR.gene-quantification.info
Good laboratory practice when performing
molecular amplification assays (QSOP 38)
Health Protection Agency (HPA),
UK; Publication Type: Standard Operating Procedures, latest
update 2010
Correction of RT-qPCR data for genomic
DNA-derived signals with ValidPrime.
Laurell H, Iacovoni JS, Abot A, Svec
D, Maoret JJ, Arnal JF, Kubista M., Nucleic Acids Res. 2012 Jan 6.
Different real-time PCR systems yield different
gene expression values.
Lu S, Smith AP, Moore D, Lee
NM., Mol Cell Probes. 2010 Oct;24(5): 315-320
An assessment of air as a source of DNA
contamination encountered when performing PCR.
Witt N, Rodger G, Vandesompele J,
Benes V, Zumla A, Rook GA, Huggett JF., J Biomol Tech. 2009
20(5): 236-240
Evaluation of different machines used to
quantify genetic modification by real-time PCR.
Allnutt TR, Ayadi M, Berben G,
Brodmann P, Lee D., J AOAC Int. 2010 93(4): 1243-1248.
Using the Taguchi method for rapid quantitative
PCR optimization with SYBR Green I.
Thanakiatkrai P, Welch L., Int
J Legal Med. 2012 126(1): 161-165
SYTO dyes and EvaGreen outperform SYBR Green in
real-time PCR.
Eischeid AC., BMC Res Notes.
2011 Jul 28;4: 263.
Troubleshooting fine-tuning procedures for qPCR
system design.
Raso
A, Mascelli S, Nozza P, Ugolotti E, Vanni I, Capra V, Biassoni
R., J Clin Lab Anal. 2011 25(6): 389-394
... ... more interesting reading about optimisation of qPCR here => optimisation.gene-quantification.info
GenEx v5 - qPCR data analysis software
GenEx
5 - A Powerful Tool For qPCR Data Analysis
GenEx is a popular software for
qPCR data processing and analysis. Built in a modular
fashion GenEx provides a multitude of functionalities for the qPCR
community, ranging from basic data editing and management to advanced
cutting-edge data analysis. View our webpage => GenEx.gene-quantification.info
Basic
data editing and management
Arguably the most important part of qPCR experiments is to pre-process
the raw data into shape for subsequent statistical analyses. The
pre-processing steps need to be performed consistently in correct order
and with confidence. GenEx Standard’s streamlined and user-friendly
interface ensures mistake-free data handling. Intuitive and powerful
presentation tools allow professional illustrations of even the most
complex experimental designs.
Advanced
cutting-edge data analysis
When you need more advanced analyses GenEx Enterprise is the product
for you. Powerful enough to demonstrate feasibility it often proves
sufficient for most users demands. Current features include parametric
and non-parametric statistical tests, Principal Component Analysis, and
Artificial Neural Networks. New features are continuously added to
GenEx with close attention to customers’ needs.
New
features
Sample handling and samples individual biology often contribute to
confounding experimental variability. By using the new nested ANOVA
feature in GenEx version 5 user will be able to evaluate variance
contributions from each step in the experimental procedure. With a good
knowledge of the variance contributions, an appropriate distribution of
experimental replicates can be selected to minimize confounding
variance and maximize the power of the experimental design! For
experiments with complex features, such as for example multifactorial
diseases, analytical relationships and classifications may not readily
be available. The support vector machine feature in the new version of
GenEx is so easy to use that it will make this advanced supervised
classification method easily available to novice users, while providing
access to advanced parameters for experts.
Download a free trail version here => GenEx.gene-quantification.info
Best regards,
Michael W. Pfaffl
responsible Editor of the Gene
Quantification Pages
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