Montag, 17. November 2014

Study Shows High Variability in Reverse Transcription Efficiency

Study Shows High Variability in Reverse Transcription Efficiency
Nov 13, 2014 via GenomeWeb

Though reverse transcription of RNA to cDNA is an essential first step for a growing number of genomics applications, researchers have long known of problems associated with RT and its effects on study results. Now, a new study has examined some of those issues and has provided a workflow and guidelines for researchers publishing RT-based data.

The study, published in Clinical Chemistry late last month, was undertaken by some of the authors of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments, or MIQE, guidelines, which debuted in 2009. It compared measures of different mRNA targets using six commercially available RT enzymes and varying sample concentrations of differing qualities. Results showed the variability was "sufficiently large to call into question the validity of many published data that rely on quantification of cDNA."
... ... read more

Freitag, 7. November 2014

Open qPCR: DNA Diagnostics for Everyone

Low Cost Real Time PCR
Turning DNA into data just became affordable, and biohacking will never be the same. Meet our open source Real-Time PCR Thermocycler.

With a design team led by one of the creators of the original OpenPCR, Chai Biotechnologies is developing a Real-Time PCR Machine with a target price of less than $1000 USD. With this, we hope to greatly expand access to the powerful technology of qPCR. The product is currently in late prototyping stages. To learn more as updates become available, subscribe to our newsletter below.

Montag, 3. November 2014

PCR Webinar Series: Troubleshooting

Dr. Tania Nolan
The Gene Team
Our sixth and final webinar in this series will be an interactive evaluation and troubleshooting of customer assays. Please send us your questions and/or failed assays and we will go through troubleshooting together to identify the problem. We will need information on the target sequence, primers and probes, how the assay was designed and QC for the primers and probes, amplification plots and melting curves (if SYBR Green/Molecular Beacon or Scorpion assay).

Donnerstag, 23. Oktober 2014

The first BDQ issue is online!

Biomolecular Detection and Quantification
Volume 1, Issue 1, September 2014

Digital PCR: A brief history
Pages 1-2
Alexander A. Morley
A current overview of commercially available nucleic acid diagnostics approaches to detect and identify human gastroenteritis pathogens
Pages 3-7
Kate Reddington, Nina Tuite, Elizabeth Minogue, Thomas Barry
Determining lower limits of detection of digital PCR assays for cancer-related gene mutations
Pages 8-22
Coren A. Milbury, Qun Zhong, Jesse Lin, Miguel Williams, Jeff Olson, Darren R. Link, Brian Hutchison
A survey of tools for the analysis of quantitative PCR (qPCR) data
Pages 23-33
Stephan Pabinger, Stefan Rödiger, Albert Kriegner, Klemens Vierlinger, Andreas Weinhäusel

Submit your manuscript

Hot papers in the field of digital PCR

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 domain. The focus of this newsletter issue is:

  • Hot papers in the field of digital PCR -
  • First event announcement -- qPCR & NGS 2015 Event -- "Advanced Molecular Diagnostics for Biomarker Discovery" -
  • "Biomolecular Detection and Quantification" -- Call for submissions -
  • GenEx 6 available - The most powerful tool for complex qPCR data analysis - download a free trial version -

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Digital PCR (dPCR)

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."

The first digital-PCR paper:
Quantitation of targets for PCR by use of limiting dilution
Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA.
Biotechniques. 1992 13(3): 444-449

We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.


More hot and new digital PCR papers =>