March 1, 2007 (Vol. 27, No. 5)
Nicholas D. Rose
Currently, real-time PCR is one of the most important techniques used in genomic analysis. The data it provides can be invaluable to determining disease mechanisms and discovering ways to target these diseases. Two new products, StepOne™ from Applied Biosystems (www.appliedbiosystems.com) and Qiagen’s (www.qiagen.com) QuantiFast™, are making this technique more accessible to a wider range of researchers, according to officials at both firms.
The StepOne system offers a low-throughput option for PCR users in a 48-well plate format. “The StepOne is designed for experiments where a 96-well plate is unnecessary, allowing you to conserve reagents and have an application-specific PCR machine,” says Rosy Lee, product manager at Applied Biosystems.
The system has an LCD touchscreen, a USB drive, and a LAN connection, which “allow researchers to monitor the StepOne from their desks,” explains Lee. “It also makes the system flexible, since it can have a PC attached but doesn’t need one.”
Low-throughput PCR offers researchers reliability and reduced in-lab time without the cost of high-throughput machines, and its optical system is able to detect fluorescence from FAM™/SYBR® Green, VIC®/JOE™, and ROX™ dyes, continues Lee.
StepOne’s software offers a wizard guide designed for first-time PCR users. “The software contains multiple wizards and tutorials, which make it easy to use. However, just because the software is easy doesn’t mean it’s not powerful,” she adds.
The QuantiFast Kit uses Qiagen’s Fast Cycling PCR technology to decrease the time required for real-time PCR. The kit was designed to work with every available real-time PCR instrument and assay, according to the company. The kit can reduce the time for a PCR run to 40–60 minutes.
The key to the improved speed is the Q-Bond additive contained in the PCR buffer, which increases DNA polymerase’s affinity for single-stranded DNA. The buffer also contains HotStarTaq Plus DNA Polymerase, which requires only five minutes, at 95°C, for activation.
Gene Expression Analysis
The availability of organism-specific primer libraries has eliminated bottlenecks associated with the use of quantitative RT-PCR to make this a cost-effective tool for gene expression analysis. To meet the high-throughput requirements for differential gene expression analysis, a team at Sigma-Aldrich(www.sigmaaldrich.com) has developed an automated method for quantitative RT-PCR using Caliper Life Sciences’ (www.caliperls.com) Sciclone ALH 3000 liquid handling workstation.
Reactions were set up by including the SYBR® Green Quantitative RT-PCR Kit and a Primer Library for Arabidopsis Pathogen-inducible Genes from Sigma-Aldrich. For these studies, total RNA was isolated from pathogen-infected Arabidopsis leaves. In a poster entitled “High Throughput Gene Expression Analysis of Arabidopsis thaliana using Quantitative RT-PCR,” Rebecca Pashia et al., presented data demonstrating the potential of quantitative-RT-PCR for expression profiling of a defined set of genes against a large number of samples.
The scientists concluded that the primer library for Arabidopsis genes is suitable for expression analysis of a defense-related study with high efficiency and reproducibility. Also, many genes included in this primer library are expressed in a pathogen-specific manner and have the potential as a disease-related gene signature for mutant characterization or other disease-related research.
The researchers noted that the walk-away automated protocol for Quantitative RT-PCR using the Primer Library for Arabidopsis Pathogen-Inducible genes represented a high-throughput platform for gene expression studies.