Applied Biosystems Reagents Improve Data Quality Of Real-Time PCR Applications

Applied Biosystems, an Applera Corporation business, today announced the release of two new application-specific master mix reagents designed to improve data quality of real-time PCR — a method used to detect and determine the amount of nucleic acids present in samples.

The TaqMan^® Gene Expression Master Mix and TaqMan^® Genotyping Master Mix reagents have been formulated to provide researchers high-quality data, resulting in a better picture of how nucleic acids (DNA, RNA) impact important biological processes. By matching the right master mix reagent — staple chemical components necessary for performing real-time PCR reactions — with the kind of application for which the reagent was designed, researchers can boost the performance of either single nucleotide polymorphism (SNP) genotyping or quantitative real-time PCR applications, compared with how these kinds of assays perform with other master mix chemistries.

Applied Biosystems developed the TaqMan Gene Expression Master Mix so that researchers can efficiently and effectively detect a broad range of nucleic acid targets in analyzed samples. When used in conjunction with TaqMan Gene Expression Assays, this new master mix reagent results in highly-sensitive assays, making it possible for researchers to detect a single copy of target nucleic acid present in a sample; even if that target is a poorly or rarely expressed gene. Use of highly-sensitive assays helps researchers to better understand how gene activity influences biological processes.

For genotyping applications, the TaqMan Genotyping Master Mix, used in conjunction with TaqMan SNP Genotyping Assays, helps researchers to accurately identify DNA sequence variations by generating data with distinct well-separated clusters of different sequence variants. They can then effectively assess how small variations in DNA sequence impact key biological processes.

For gene expression studies, and other quantitative real-time PCR applications, the TaqMan Gene Expression Master Mix helps researchers to effectively compare changes in the expression levels of genes from a wide variety of samples, including diseased and healthy tissues, which requires efficient use of limited quantities of starting sample. Researchers can also accurately differentiate between targets of similar DNA or RNA sequences, and detect two separate DNA targets in the same tube, which increases the number of samples that can be processed in a single experiment.

“The development of these master mixes with new enzymes demonstrates Applied Biosystems’ continued strategic focus on bringing high-quality, application-specific solutions for real-time PCR to market,” said Mark Stevenson, president for Applied Biosystems’ molecular and cell biology division. “Our customers are continuing to derive new applications from their real-time PCR instrument systems; therefore, it is important that we continue to play a leading role in providing them with differentiated consumables that are tailored to their specific scientific objectives.”

Researcher at Trinity College Detects Many Genes in Limited Amounts of RNA Samples
In gene expression studies of thyroid cancer, Dr. Orla Sheils, a senior lecturer specializing in molecular histopathology at Trinity College in Dublin, Ireland, often works with archived tissue samples. The storage process of embedding these samples in wax and freezing them typically degrades nucleic acids, leaving just a few copies of compromised RNA available to analyze. When she tested the TaqMan Gene Expression Master Mix, including it as part of an assay she used in the analysis of thyroid cancer tissue samples, Dr. Sheils found that gene expression patterns from archived samples correlated well with those generated from fresh material.

“The TaqMan Gene Expression Master Mix allows us to detect more genes in limited samples,” said Sheils. “Our need for the master mix was a combination of the sensitivity of the assay and getting the maximum value for the amount of material that we have. So, by using this new master mix, we can now further stretch the limited amount of RNA that we have from patient samples. We can get a lot more information from a smaller amount of sample.”

Pasteur Institute Obtains High-Quality SNP Genotyping Data for Diabetes Research
For genotyping studies, such as allelic discrimination experiments, researchers often aim to associate small variations in genomic DNA with a particular trait or disease. These kinds of studies help researchers to better understand how genetic variation impacts key biological processes. For example, Emmanuel Vaillant, an engineer at the Institute of Biology of Lille, Pasteur Institute in France, recently tested the TaqMan Genotyping Master Mix, using it in assays for genotyping studies that explore how SNPs in genes contribute to breakdowns in biological processes associated with diabetes. As a result, he was able to generate high-resolution clusters of alleles in graphical data plots.

“In a SNP genotyping application for a population study, we were not able to obtain satisfactory results until I used the new TaqMan Genotyping Master Mix,” said Vaillant. “After performing the assay with this master mix, we obtained exceptional data with discrete clusters of alleles displayed in the graphical plot.”

Both of these new master mix reagents contain a highly-purified enzyme as well as all necessary components —excluding template, primers, and probes — needed to perform TaqMan-based real-time PCR applications. The enzyme minimizes non-specific product formation and allows reactions to be started at room temperature, which contributes to more effective workflows. For high-throughput, real-time PCR applications, researchers can employ automated handling systems as both master mixes remain stable in the reaction mixture, even after 72 hours on the bench-top.

Each of these new master mix reagents can easily be incorporated into existing application protocols, without changing thermal cycling conditions. The application-specific master mix reagents can be used with more than 700,000 Applied Biosystems pre-designed TaqMan Gene Expression Assays, over 4 million TaqMan^® SNP Genotyping Assays, or any of the custom TaqMan Assays that are available for customer-specific gene expression or genotyping assays.