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Archive for the ‘Bio-Rad Product Highlight’ Category

Bio-Rad Sponsors New Version of MIQE qPCR App for Real-Time PCR and Digital PCR

 :: Posted by American Biotechnologist on 08-28-2013

miqe appBio-Rad Laboratories is sponsoring a new version of the MIQE qPCR app. Researchers can use the new version to validate their digital PCR (dPCR) experiments according to the recently published digital MIQE (dMIQE) guidelines (Huggett et al. 2013).

Professors Michael Pfaffl (TU Munich, Weihenstephan, Germany) and Afif Nour (King Abdulaziz University, Saudi Arabia) designed the original MIQE qPCR app to help researchers improve their real-time PCR (qPCR) assay protocols by making it easier to adopt a set of best practices described in an earlier publication: The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments (Bustin et al. 2009). Progress bars in the app show the percentage of assay compliance as each item in the MIQE checklist is completed.

“We were pleased to find that the MIQE qPCR app encouraged our customers to follow the new MIQE guidelines,” said Jean-Pierre Dakkak, a laboratory equipment trading company manager.

“Researchers are really eager to learn how this app can make their life easier,” said Dr. Nour. “They report it instills confidence in the validity of every qPCR or dPCR project.”

The new updates include:

  • Project-specific checklists — checklists remove unnecessary items; for example, reverse transcription items are irrelevant in DNA research
  • Updated references — researchers can stay current with the latest MIQE and dMIQE literature and qPCR symposiums
  • Easy export — users can save their projects and share them with colleagues

The MIQE qPCR app runs on the Apple iPhone, iPod touch, and iPad. It has been downloaded more than 6,500 times in 87 countries. To get your copy, visit http://bit.ly/MIQEapp.

Droplet Digital™ PCR provides accurate quantification of next-generation sequencing libraries

 :: Posted by American Biotechnologist on 08-19-2013

A study published today found that Droplet Digital PCR (ddPCR™) can be used as an accurate and precise method for quality control of next-generation sequencing (NGS) libraries. NGS library QC is essential to optimizing sequencing data yield, thereby increasing efficiency and throughput while lowering cost. The research was published in the in the August issue of Biotechniques.

“While real-time PCR has traditionally been used to quantify libraries, we determined that the only truly accurate way to reproducibly quantify our NGS libraries is with ddPCR,” said Dr. Jason Bielas, lead author and Assistant Member in the Public Health Sciences Division at Fred Hutchinson Cancer Research Center in Seattle, Wash.

Quantifying NGS Libraries and Why It Matters

Various commercial NGS technologies require users to load a precise number of viable DNA library molecules onto the instrument to optimize data yield. Performing a sequencing run with either too many or too few library molecules results in compromised data and sometimes no data at all – wasting sample, expensive reagents, user time, and instrument time.

Moreover, fewer bases might be sequenced if library molecules are not the appropriate length to fully utilize the sequencing platform, thus limiting throughput. Given this, quantifying library molecules and determining fragment size range have become crucial steps in library preparation.

NGS instrument manufacturers recommend quantifying libraries using real-time quantitative PCR (qPCR) and determining their size range using gel or capillary electrophoresis. Each of these has its limitations, though, and the steps recommended to address them, can be time-consuming and expensive.

Advantages of ddPCR for Quantifying NGS Libraries

To simultaneously quantify and determine the size distribution of target DNA with a single ddPCR assay, Dr. Bielas and his team exploited a relationship between droplet fluorescence and amplicon size. They confirmed the accuracy and precision of this method by applying it to NGS library preparation.

The ddPCR assay they designed – known as QuantiSize – was developed using the QX100 ddPCR system from Bio-Rad Laboratories. QuantiSize offers the ability to determine the absolute quantity and the detailed size distribution of target DNA in a single ddPCR reaction well, thus avoiding the drawbacks of other independent quantification and size determination methods.

“Now that we have discovered this new correlation, we can also use ddPCR to extract more information on the characteristics of DNA based on the range of fluorescence that can occur within each droplet,” said Bielas.

Having demonstrated the efficacy of this technique, Dr. Bielas is now planning to leverage the relationship between ddPCR fluorescence and amplicon size to explore mutagenic deletion events in both the human nuclear and mitochondrial genomes.

Bio-Rad News: New biomarker could reveal Alzheimer’s disease years before onset

 :: Posted by American Biotechnologist on 08-14-2013

A study published today reported the identification of what may be the earliest known biomarker associated with the risk of developing Alzheimer’s disease (AD). The results suggest that this novel potential biomarker is present in cerebral spinal fluid (CSF) at least a decade before signs of dementia manifest.

“If our initial findings can be replicated by other laboratories, the results will change the way we currently think about the causes of Alzheimer’s disease,” said Dr. Ramon Trullas, research professor at the CSIC Institute of Biomedical Research of Barcelona and lead author of the study that was published in Annals of Neurology. “This discovery may enable us to search for more effective treatments that can be administered during the preclinical stage.”

Difficult Diagnosis

Alzheimer’s disease affects more than five million Americans and is the sixth leading cause of death in the United States. At present, the only way to accurately diagnose the disease is by post-mortem neuropathological analysis. The relationship of currently known biomarkers with the cause of the disease is unclear, making it nearly impossible to diagnose preclinical stages of the disease with any real certainty.

The CSIC researchers demonstrated that a decrease in the content of mitochondrial DNA (mtDNA) in CSF may be a preclinical indicator for Alzheimer’s disease; furthermore, there may be a directly causative relationship. The hypothesis is that decreased mtDNA levels in CSF reflect the diminished ability of mitochondria to power the brain’s neurons, triggering their death. The decrease in the concentration of mtDNA precedes the appearance of well-known biochemical Alzheimer’s biomarkers (the Aβ1-42, t-tau, and p-tau proteins), suggesting that the pathophysiological process of Alzheimer’s disease starts earlier than previously thought and that mtDNA depletion may be one of the earliest predictors for the disease.

In addition to enabling an investigation of the potential causal relationship of mtDNA and Alzheimer’s progression, the use of mtDNA as an index of preclinical Alzheimer’s disease provides an important advantage over previous biochemical markers: the detection of this novel nucleic acid biomarker is unhampered by the technical difficulties associated with protein detection. mtDNA can be readily quantified by real-time quantitative PCR (qPCR) or droplet digital PCR (ddPCR).

Quantitation of mtDNA

Prior to this study, researchers had not reported that circulating cell-free mtDNA could be detected in human CSF. But with this study, Dr. Trullas’ team was able to both detect and reproducibly quantitate mtDNA using qPCR, carefully optimized by adhering to the MIQE guidelines.

To validate their qPCR findings, Dr. Trullas’ team used Bio-Rad Laboratories’ QX100™ Droplet Digital™ PCR system. Unlike qPCR assays, the QX100 system provides an absolute quantification of target DNA molecules without the need for a standard curve. In addition, an important factor for their CSF analysis was that the Droplet Digital PCR system did not require sample purification to remove PCR inhibitors, as is necessary for qPCR assays.

“Droplet Digital PCR allowed us to validate our initial qPCR measurements because it provides absolute quantitation at the single-molecule level without relying on a standard curve,” said Dr. Trullas. “As the technology becomes more widely adopted, we anticipate that Droplet Digital PCR will be the future of detecting mtDNA in cerebral spinal fluid.”

Dr. Trullas hopes that other laboratories and hospitals will successfully replicate his group’s research results, confirming that reduced mtDNA levels should be investigated as a possible cause of Alzheimer’s disease. By finding a way to block this degeneration, clinicians may be able to diagnose and treat Alzheimer’s disease before symptoms appear.

The Fastest Gel in the Lab

 :: Posted by American Biotechnologist on 08-08-2013

New PrimePCR™ Probe Assays for Human and Mouse Genomes

 :: Posted by American Biotechnologist on 07-02-2013

Bio-Rad Laboratories, Inc. today announced the launch of its PrimePCR probe assays for quantitative real-time PCR (qPCR). The new assays for human and mouse genomes set the bar for specificity, sensitivity, and linear dynamic range.

PrimePCRBio-Rad has again partnered with Biogazelle, a qPCR data analysis and services company, to design probe assays for qPCR gene expression analysis. The fluorescently labeled oligonucleotide probes were designed to complement Bio-Rad’s high-specificity PCR primer assays, enabling multiplex assays and eliminating the need for melt curve analysis.

The primer pair of each probe assay has been wet-lab validated following strict assay performance standards for amplification efficiency, specificity, sensitivity, and linear dynamic range. These specifications help researchers meet the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines.

The new probe assays are validated for use with iScript™ advanced cDNA synthesis kit for RT-qPCR and SsoAdvanced™ universal probes supermix for superior performance.

The assays are available with probes labeled with FAM, HEX, or TEX 615 fluorophores, and each includes a gene-specific synthetic qPCR template designed as a positive control. The probe assays can be used with CFX real-time PCR detection systems and data analysis software, streamlining PCR data collection and analysis.

PrimePCR probe assays are available in 500, 1,000, and 2,500 reaction sizes.

For more information on Bio-Rad’s PrimePCR products, please visit www.bio-rad.com/PrimePCR.