qPCR NEWS - August 2012 - focus on single-cell qPCR

Editor www.Gene-Quantification.info via methods%40net.bio.net (by editor from gene-quantification.info)
Thu Aug 16 08:48:54 EST 2012


qPCR NEWS - August 2012 - focus on single-cell qPCR
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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=20
Gene Quantification homepage. The focus of this newsletter issue is:

* UPDATE of various new papers using single-cell PCR technologies in qPCR a=
nd NGS  -  http://singlecell.Gene-Quantification.info
* Second announcement qPCR & NGS Symposium in March 2013 - http://www.qPCR-=
NGS-2013.net=20
* GenEx - a powerful tool for qPCR data analysis - download a free trial ve=
rsion  -  http://GenEx.gene-quantification.info

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If this newsletter is not displayed correctly by your email client, please =
use following http://qPCRnews.gene-quantification.info
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UPDATE of various new papers using single-cell PCR technologies in qPCR and=
 NGS
=3D>  http://singlecell.Gene-Quantification.info

Single-cell molecular-biology is a relatively new scientific branch in biol=
ogy. The first single-cell analysis were involved in the characterization o=
f mitochondrial DNA in 1988. Single-cell DNA analysis, in particular genomi=
c DNA, is important and may be informative in the analysis of genetics of c=
ell clonality, genetic anticipation and single-cell DNA polymorphisms. Nowa=
days for most sceientists the quantitative transcriptomics in a single-cell=
 is much more important, and the analytical method of choice is the quantit=
ative real-time RT-PCR. In single-cell biology the absolute abundance of pa=
rticular mRNAs or microRNAs and their up- or down-regulation in a single ce=
ll, compared to their neighbour cells, is the goal. The need for quantitati=
ve single-cell mRNA analysis is evident given the vast cellular heterogenei=
ty of all tissue cells and the inability of conventional RNA methods, like =
northern blotting, RNAse protection assay or classical block RT-PCR, to dis=
tinguish individual cellular contributions to mRNA abundance differences.=
=20
  =20
new papers Method of the year - Methods to watch - Special feature
Single-cell methods - Improved single-cell methods are helping to unravel b=
iological complexity
We present important methods and areas of methodological development worth =
watching in the coming years.
Nature Methods - VOL.9 NO.1 - JANUARY 2012 - 35

The heterogeneity of cells in culture and in organisms poses a challenge fo=
r many experi-mental measurements. Population measure-ments are necessarily=
 averages, masking the behavior of minority subpopulations and effectively =
blinding researchers to possibly interesting differences between cells.The =
alternative is to make measure-ments on single cells. Methodologically spea=
king, this, too, is challenging on sever-al fronts. Molecular analyses, whe=
ther on a particular macromolecule or at an =91omic=92 scale, can be diffic=
ult (or even impossible) to accomplish on the amount of mate-rial extracted=
 from one cell. Methods with increased sensitivity are therefore in demand.=
 Throughput is also a bottle-neck. Basing firm conclusions on single-cell m=
easurements means that one must be able to quickly and accurately analyze m=
any cells. Finally, it is often necessary to analyze single cells in a mul-=
tiplexed fash-ion, either because the cells exist in a heteroge-neous pop-u=
lation or because one wants to measure many parameters at the same time.The=
re continue to be methodologi-cal advances on all of these fronts. Mass cyt=
ometry, for instance - in which iso-topes are used as antibody labels inste=
ad of fluorescent probes=97considerably extends the multiplexing capabiliti=
es of flow cytometry (Science 332, 687=96695; 2011).   Is the measurement o=
f gene expression,  digital reverse-transcriptase PCR in a microfluidics de=
vice makes it possible to simultaneously monitor the expres-sion of hundred=
s of genes in hundreds of single cells. As demonstrated in a recent study o=
f tumor heterogeneity, this can be combined with single cell sorting and wi=
th statistical clustering methods to begin to dissect the cellular subpopul=
ations that constitute a tissue (Nat. Biotechnol. 29, 1120=961127; 2011).

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Cell Biology. Using cell-to-cell variability - a new era in molecular biolo=
gy
Pelkmans L.
Science. 2012 Apr 27;336(6080): 425-426

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Genomic analysis at the single-cell level
Kalisky T, Blainey P, Quake SR.
Annu Rev Genet. 2011;45: 431-445

Studying complex biological systems such as a developing embryo, a tumor, o=
r a microbial ecosystem often involves understanding the behavior and heter=
ogeneity of the individual cells that constitute the system and their inter=
actions. In this review, we discuss a variety of approaches to single-cell =
genomic analysis.

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A single molecule view of gene expression
Larson DR, Singer RH, Zenklusen D.
Trends Cell Biol. 2009 (11): 630-637

Analyzing the expression of single genes in single cells appears minimalist=
ic in comparison to gene expression studies based on more global approaches=
. However, stimulated by advances in imaging technologies, single-cell stud=
ies have become an essential tool in understanding the rules that govern ge=
ne expression. This quantitative view of single-cell gene expression is bas=
ed on counting mRNAs in single cells, monitoring transcription in real time=
, and visualizing single proteins. Parallel advances in mathematical models=
 based on stochastic, discrete descriptions of biochemical processes have p=
rovided crucial insights into the underlying cellular mechanisms that contr=
ol expression. The view that has emerged is rooted in a probabilistic under=
standing of cellular processes that quantitatively explains both the mean a=
nd the variation observed in gene-expression patterns among single cells. T=
hus, the close coupling between imaging and mathematical theory has establi=
shed single-cell analysis as an essential branch of systems biology.

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Single-cell gene-expression profiling and its potential diagnostic applicat=
ions
Stahlberg A, Kubista M, Aman P.
Expert Rev Mol Diagn. 2011 (7): 735-740

Gene-expression profiling has been successfully applied in various diagnost=
ic applications, but its full capacity is yet to be realized. Samples are g=
enerally prepared from a mixture of different cells that are present in unk=
nown proportions. Cells are, in many aspects, unique in their characteristi=
cs and this heterogeneity confounds the expression profile. The development=
 of new and robust techniques to measure gene expression in single cells op=
ens new avenues in molecular medicine. Today, gene-expression profiles of i=
ndividual cells can be measured with high precision and accuracy, identifyi=
ng different cell types as well as revealing heterogeneity among cells of t=
he same kind. Here, we review practical aspects of single-cell gene-express=
ion profiling using reverse transcription quantitative real-time PCR and it=
s potential use in diagnostics.=20


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New papers using single-cell PCR technologies in qPCR and NGS  -  http://si=
nglecell.Gene-Quantification.info

- Multimarker gene analysis of circulating tumor cells in pancreatic cancer=
 patients: a feasibility study
- Mammalian genes are transcribed with widely different bursting kinetics
- Measuring single-cell gene expression dynamics in bacteria using fluoresc=
ence time-lapse microscopy
- Quantification noise in single cell experiments
- Identifying single-cell molecular programs by stochastic profiling
- High-throughput microfluidic single-cell RT-qPCR
- RNA-Seq analysis to capture the transcriptome landscape of a single cell
- Development and applications of single-cell transcriptome analysis
- Quantitative RT-PCR gene expression analysis of laser microdissected tiss=
ue samples
- mRNA and microRNA expression profiles in circulating tumor cells and prim=
ary tumors of metastatic breast cancer patients
- Comprehensive qPCR profiling of gene expression in single neuronal cells
- Single cell transcriptomics of neighboring hyphae of Aspergillus niger
- RT-qPCR based quantitative analysis of gene expression in single bacteria=
l cells
- Circulating tumor cells in breast cancer: detection systems, molecular ch=
aracterization, and future challenges
- Molecular characterization of circulating tumor cells in breast cancer: c=
hallenges and promises for individualized cancer treatment
- Gene expression profile of circulating tumor cells in breast cancer by RT=
-qPCR
- Single-cell gene-expression profiling reveals qualitatively distinct CD8 =
T cells elicited by different gene-based vaccines
- High throughput single cell expression profiling: Taking a closerlook on =
biological response
- Quantification of circulating endothelial and progenitor cells: compariso=
n of quantitative PCR and four-channel flow cytometry
- Parthenogenic blastocysts derived from cumulus-free in vitro matured huma=
n oocytes


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Second announcement qPCR & NGS Symposium in Freising-Weihenstephan
18-22 March 2013
http://www.qPCR-NGS-2013.net
Download the latest Event Flyer =3D> http://tinyurl.com/qPCR-NGS-2013

On behalf of the Organisation Committee and the Scientific Board it is a gr=
eat pleasure to invite you to the 6th International qPCR & NGS 2013 Event. =
The event is divided in a 3-day scientific Symposium with an Industrial Exh=
ibition and various 2-day Application Workshop to be held at the Center of =
Life Science in Freising Weihenstephan, Technische Universit=E4t M=FCnchen =
(Germany). The great international interest in the previous meetings ( qPCR=
 2004 to qPCR 2011 ) led us to the decision to repeat the Symposium in spri=
ng 2013. We expect 500-600 participants coming from all over the world, in =
2011 we could welcome participants from 56 contries, and roughly 30-40 inte=
rnational companies in the qPCR Industrial Exhibition.

We have set the date for the qPCR & NGS 2013 Event to 18th - 22nd March 201=
3. The event location is the central lecture hall complex and the foyer at =
TUM (Technical University of Munich) in Freising Weihenstephan, Germany (Go=
ogle Maps link or Google Earth link). The TUM and the Biotech region around=
 Munich is part of the largest Biotech cluster in Europe, located close to =
the Munich airport in the heart of Bavaria.

The focus of the qPCR & NGS 2013 Event will be on:   Next Generation Thinki=
ng in Molecular Diagnostics

Leading academic researchers and industrial contributors in the field will =
participate in the symposium, which will be an arena for fruitful discussio=
ns between researchers of different backgrounds. The Symposium Talks, Poste=
r Sessions, Industrial Exhibition and associated qPCR & NGS Application Wor=
kshops offer an overview of the present knowledge and future developments i=
n qPCR, next generation sequencing and gene expression measurement technolo=
gy and its wide applications in research.

The symposium will focus on 70 lectures and a huge poster exhibition will b=
e presented by internationally recognised experts in their field. The empha=
sis will be on unbiased, didactic information exchange. Internationally ren=
own speakers will be participating in a lively and exciting programme enabl=
ing the valuable exchange of information in the qPCR and Next Generation Se=
quencing field. One third of the talks will be presented by selected invite=
d speakers, one third will be selected from the submitted abstracts and one=
 third will be presented by qPCR & NGS related company R&D representatives.=
 All scientific contributions will be published in the Symposium Proceeding=
s (ISBN to be announced).

Full papers from selected invited academic and industrial speakers and appl=
ication notes from industrial speakers will be published in a METHODS speci=
al issue =93Transcriptional Biomarkers=94  edited by Michael W. Pfaffl (pub=
lished January 2013).  At the meeting all participants will get  a free har=
d cover of this special Methods issue. Please have a look to our previous i=
ssue  =3D> =93The ongoing evolution of qPCR=94 METHODS special qPCR Vol 50 =
issue 4  (April 2010)

Please register using the Internet based ConfTool registration and submissi=
on platform =3D> http://registration.qPCR-NGS-2013.net

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Symposium Talk and Poster sessions:
http://sessions.qpcr-ngs-2013.net/

- Main Topic:   Molecular diagnostics
- Main Topic:   Next Generation Sequencing  (NGS)
- Main Topic:   Transcriptional Biomarkers
- High throughput analysis in qPCR
- Systems biology
- Single-cells diagnostics
- MIQE & QM strategies in qPCR
- non-coding RNAs - microRNA, siRNA, long non-coding RNAs
- Digital PCR  &  Nano-fluidics
- Pre-analytical Steps
- BioStatistics & BioInformatics
- qPCR & NGS data analysis
- Lunch Seminars:
   - qBASEplus - data analysis lunch seminar
   - GenEx - data analysis lunch seminar
   - NGS data analysis lunch seminars
   - more to be announced........


View our qPCR 2011 event trailer on YouTube =3D> http://www.youtube.com/wat=
ch?v=3Dcp8WwPyLW8Y

Download the latest Event Flyer =3D> http://tinyurl.com/qPCR-NGS-2013

Please register using the Internet based ConfTool registration and submissi=
on platform =3D> http://registration.qPCR-NGS-2013.net

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GenEx 5 -  A Powerful Tool For qPCR Data Analysis
Download a free trail version here =3D> http://GenEx.gene-quantification.in=
fo

GenEx is a popular software for qPCR data processing and analysis. Built in=
 a modular fashion GenEx provides a multitude of functionalities for the qP=
CR community, ranging from basic data editing and management to advanced cu=
tting-edge data analysis. View our webpage =3D> http://GenEx.gene-quantific=
ation.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 confiden=
ce. GenEx Standard=92s streamlined and user-friendly interface ensures mist=
ake-free data handling. Intuitive and powerful presentation tools allow pro=
fessional 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 yo=
u. Powerful enough to demonstrate feasibility it often proves sufficient fo=
r most users demands. Current features include parametric and non-parametri=
c statistical tests, Principal Component Analysis, and Artificial Neural Ne=
tworks. New features are continuously added to GenEx with close attention t=
o customers=92 needs.

New features
Sample handling and samples individual biology often contribute to confound=
ing experimental variability. By using the new nested ANOVA feature in GenE=
x version 5 user will be able to evaluate variance contributions from each =
step in the experimental procedure. With a good knowledge of the variance c=
ontributions, an appropriate distribution of experimental replicates can be=
 selected to minimize confounding variance and maximize the power of the ex=
perimental design! For experiments with complex features, such as for examp=
le multifactorial diseases, analytical relationships and classifications ma=
y not readily be available. The support vector machine feature in the new v=
ersion of GenEx is so easy to use that it will make this advanced supervise=
d classification method easily available to novice users, while providing a=
ccess to advanced parameters for experts.

Download a free trail version here =3D> http://GenEx.gene-quantification.in=
fo

GenEx PDF user guides:
* GenEx user guide=20
* GenEx user guide - Exiqon Wizard=20
* GenEx user guide - Roche Wizard


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 to further scientists and friends who are interested in qPCR !

Best regards,
Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages


If this newsletter is not displayed correctly by your email client, please =
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