Turmoil and massive layoffs in the auto industry are one indication that it’s a great time to be in life sciences. But
schadenfreude need not be the only impetus for merriment in your work. You will achieve such bliss once you discover the ease and reliability of the latest real-time PCR reagents and kits. Companies have clearly listened to your woes—and have replied with solutions to improve your DNA-counting ability.
Any of the problems with real-time PCR, also called quantitative PCR (qPCR), probably pale in comparison with the tedium and inaccuracies of other gene quantification methods. Northern blot analyses and RNase protection and S1 nuclease mapping assays were tedious to perform and fraught with approximations. In addition to its frustration with the inability to scale-up these techniques for high-throughput analysis, the field was hungry for more robust methods. The paper “Simultaneous amplification and detection of specific DNA sequences,” published in the April 1992 issue of the journal Nature Biotechnology, satiated this appetite.
The basic premise described in that first paper guides the practice of today: The amount of light collected from PCR reaction products can be used to calculate the number of templates that were present at the start. Now the favored method for quantifying mRNA, DNA, microRNA, or other species of nucleic acid strands, qPCR has gained a large following and spawned a whole line of products. You can find reagents and kits for general quantification or those specifically designed for the “analysis of differential mRNA expression and SNP detection, including splice variant discrimination, pathogen load diagnosis, cancer marker quantification and genotyping,” according to a paper published in the July 2006 issue of Nucleic Acids Research.
You’ll find that kits make life a lot easier. Most companies develop kits with pre-optimized reagents to save you the hassle of setting the perfect reaction conditions and cycling parameters. While some kits are designed for specific instruments and brands, you will also find those that work just as well on any instrument. If your instrument is built with a halogen lamp, you can opt for kits that include the ROX reference dye. Or, you may be content with SYBR Green and the other fluorescent dyes that have become so pervasive. The variety of colors has given birth to kits for multiplexing, which allow you to simultaneously chart the amplification of multiple starting targets. Some kits can achieve this even when the amounts of starting targets differ by up to 100,000-fold.
No need to worry about low abundance samples—you will find many kits that can detect as few as 10 copies of starting target. Reaction mixes also contain ingredients to overcome some of the most frustrating problems, such as the formation of non-specific products and primer-dimers, the introduction of contaminating DNA and unstable annealing of primers with the target template. You will find kits that include the ability to alert you to the presence of inhibitory substances, as well—substances such as phenol, ethanol, guanidine, and EDTA.
The list below contains some of the latest products to enter the market. They’re all designed to carry you far—without churning greenhouse gases into the warming atmosphere.
The SYBR® GreenER™ qPCR reagent system incorporates the latest technology to produce the most reliable gene expression data from real-time qPCR. The SYBR® GreenER™ system is specially formulated to offer the best sensitivity, specificity, and reproducibility. Compared to SYBR® Green dye, this novel dye produces a brighter signal and significantly reduced PCR inhibition and exhibits nearly identical spectral characteristics. Use the SYBR® GreenER™ qPCR reagent system just like your current SYBR® Green dye without changing instrument settings or filters, and achieve superior performance.
Our new Alien® QRT-PCR Inhibitor Alert is an in vitro transcribed RNA that is non-homologous to the sequences currently available in GenBank. It can be used as an external RNA control for detection of potential QRT-PCR inhibitory substances in your RNA samples such as phenol, ethanol, guanidine, heparin, and EDTA. Furthermore, the Alien QRT-PCR inhibitor alert can also be an ideal reference for assay standardization to allow data comparisons from multiple experiments, across platforms, and between laboratories.
Sensitivity without Compromise – Power SYBR® Green PCR Master Mix delivers highly sensitive and reproducible DNA, cDNA, and RNA quantitation. It detects as few as two copies of a target gene over a wide dynamic range of template concentrations without sacrificing important performance parameters such as specificity and data uniformity. Whether you are doing gene expression, pathogen detection, or target validation, the newly optimized formulation minimizes variation, ensuring consistency between experiments.
Dual-labeled TaqMan® probes are highly valuable tools because they are designed to pair to a unique target, providing sequence specific detection which can not be obtained with DNA intercalating dyes such as SYBR Green. FAM-BHQ® labeled probes have become the standard for real-time qPCR assays due to their brightness, high signal to noise and compatibility with all major thermocyclers. Biosearch offers a unique HPLC purified FAM-BHQ probe, the ValuProbe™, for budget conscience users who may be transitioning from SYBR green or optimizing and screening multiple assays. Each ValuProbe provides enough product to run approximately 1000 reactions.
Roche’s FastStart SYBR Green Master is a ready-to-use, 2x-concentrated master mix for qPCR and two-step qRT-PCR assays. This convenient master mix is based on a hot-start DNA polymerase which eliminates the formation of primer-dimers and nonspecific products during qPCR reaction setup. The unique buffer system removes the need to titrate MgCl2, ensuring robust assay performance with minimal reagent optimization. The FastStart SYBR Green Master is available in 5 ml or 50 ml kits, with or without ROX normalization dye.
Take real-time, one-step RT-PCR to the next level with QuantiTect® Multiplex RT-PCR Kits, which make multiplex analysis absolutely simple. Up to 5 RNA targets in one tube or well can be reliably quantified without any tedious optimization steps. The kits are designed for gene expression analysis, providing optimal results even in multiplex reactions to quantify transcripts that differ greatly in abundance. Target genes and the endogenous control are coamplified with similar efficiencies to ensure highly precise relative quantification.
