As the sequence of the human genome is deciphered, determining gene function and gene expression are becoming the focus of life science research. Carola Schade and Achim Wehren reports.
Various high throughput technologies (for example, quantitative polymerase chain reaction (PCR), NASBA, and microarray-analysis) are being used to determine gene expression profiles. These techniques demand purification technologies and instrumentation that offer high levels of reproducibility, sensitivity, and high throughput of samples.
Real-time quantitative PCR uses fluorescent chemistries and photometric detection systems to monitor the accumulation of product during the PCR. The number of PCR-cycles taken to detect fluorescence is directly related to the number of target nucleic acid molecules and allows determination of the initial number of target sequences.
When coupled to a carefully controlled reverse transcription (RT) reaction, which transcribes complementary DNA (cDNA) from mRNA, this powerful technique can be used to measure levels of messenger RNA (mRNA) transcribed from a gene. Instruments used to automate this technique became available in the late 1990s. Use of these instruments has meant a dramatic increase in throughput capabilities (up to 5000 sample-wells per day) due to parallel processing of large numbers of samples, multiplexing, and highly sensitive detection technologies.
However, quality of the prepared sample is vital for accuracy of downstream results, as any inconsistencies in sample preparation will be amplified in subsequent steps. It follows that life-scientists require reliable purification technologies to match these high-throughput analytical techniques.
The Qiagen BioRobot 9604 (Fig. 1) is an automated system developed for laboratories requiring purification of high-quality RNA from animal and human cells. In particular, the Qiagen BioRobot suits high-throughput gene-expression analysis, including real-time quantitative PCR.
The robotic workstation is designed to maximise reproducibility for all sample preparation steps (eg, cell lysis, RNA purification, reaction setup), ensuring reliable results in subsequent analysis. Optimised, ready-to-run protocols allow RNA from up to 192 samples to be purified in 90 minutes, using the RNeasy 96 technology.
Reliable automated purification
The RNeasy 96 BioRobot procedure provides rapid, high-throughput, isolation of RNA from 10 to 5 x 105 animal or human cells. RNeasy technology combines guanidine-isothiocyanate (GITC) mediated lysis and inactivation of nucleases with the speed of silica-gelmembrane purification.
After positive identification of individual 96-well cell-culture plates using the BioRobot Labware Identification System, the RNeasy BioRobot procedure starts with removal of cell culture medium. Cell lysis is performed in the culture vessel, using the integrated shaker system under the highly denaturing conditions of GITC.
Ethanol is added to provide appropriate binding conditions, and samples are transferred to the RNeasy 96modules located in the vacuum manifold top plates. Vacuum is applied to the lysate, drawing it through the membrane disks; total RNA is bound and contaminants are efficiently washed away. High-quality RNA is then eluted in a small volume of RNAse-free water, ready for use in downstream applications.
Two computer-controlled vacuum manifolds are integrated into the BioRobot worktable, ensuring consistent processing the of 2 x 96 samples.
RNA yields are very high and extremely reproducible, because of the the optimised chemistries, elimination of alcohol precipitation, and standardised automated processing.
RNA purified using RNeasy 96 technology on the BioRobot 9604 can be used directly for TaqMan analysis. Threshold-value CVs of less than three per cent are consistently observed in TaqMan analysis using BioRobot purified RNA from identical, independently prepared, samples. A ready to run protocol template for TaqMan reaction setup is provided with the BioRobot 9604. This template can be easily modified using QIAsoft, the BioRobot operating software, to suit different user requirements. Variable amounts of template, varying template positions, and numbers of primers (eg, for chequer board reaction setup) are easily accommodated using table functions integrated into the software.
Cross-contamination free pipetting is of special importance for sensitive applications such as TaqMan reaction setup, or when purifying RNA from more than one sample type, and is ensured by specially designed disposable tips and tip-change system. Filter barriers within the tips prevent cross contamination by aerosols.
The QIAsoft process documentation system tracks and reports the progress of each sample throughout all procedures. A report file is automatically generated, saved, and printed at the end of each run for archiving in compliance with GLP requirements. Sample and process information are easily exchanged with other laboratory instruments, including all commonly used computer systems and laboratory information management systems (LIMS), providing complete documentation of overlapping or consecutive processes. This allows the BioRobot 9604 to be easily integrated into existing laboratory environments, and facilitates the flow of information between individual laboratories or research groups.
Conclusion
Automated protocols using RNeasy purification technology and the BioRobot 9604 allow fast, reliable isolation of total RNA. The combination of standardised processing, accurate liquid handling, and efficient recovery of high-quality RNA provides highly reproducible results. Purified RNA performs excellently in downstream analysis and typically gives CVs of less than three per cent in TaqMan analysis. Speed, reliability, and high throughput suits the BioRobot 9604 to large-scale gene-expression projects.
ENQUIRY No 87
Carola Schade and Achim Wehren are with Qiagen GmbH, Hilden, Germany. www.qiagen.com
