Nucleic acid-based technologies: Application amplified

TATAA Biocenter AB, Gothenburg, Sweden. .
Pharmacogenomics (Impact Factor: 3.22). 11/2004; 5(7):767-73. DOI: 10.1517/14622416.5.7.767
Source: PubMed


The meeting was excellent, covering all the steps of gene expression analysis, as well as considerations on high-throughput techniques and examples of important applications. Clearly, what is most important for successful analysis is the quality of the sample material. When studying fixed samples, extracting good-quality RNA becomes an issue. DSP fixation seems to preserve RNA better than formalin and ethanol, and random priming works better than poly(dT) when transcribing the RNA of poor quality. Any freshly prepared RNA is rapidly degraded if RNases are not inactivated. This can be done by salting out or adding storing solution, such as RNeasy from Qiagen [6]. For matrices of low complexity, the cell-to-signal system from Ambion [7] lyses cells and is compatible with both RT and PCR. Still another approach is to use filters, such as those developed by Whatman [8], to purify and store nucleic acids. Frequently, the collected material must be amplified for analysis. For genome analysis, WGA, as developed by molecular staging [8], is an option. Other approaches are based on fragmenting the genome and adding adapters to it for PCR amplification and subsequent microarray analysis for either SNPs, as developed by Perlegen [9] and Parallel [10], or massive parallel sequencing, as developed by 454 Life Sciences [11]. If the DNA is damaged, an option may be to use the DNA polymerase repair enzyme blend called Restorase from Sigma-Aldrich [12]. For direct parallel analysis of RNA, Genentech's [13] NACA can be used. Alternatively, RNA can be amplified using T7 amplification, or a more advanced variant, such as Ovation from NuGEN [14], could be employed. These methods introduce some bias in the expression pattern, but are good enough for most purposes. The RNA can be analyzed en masse by microarray hybridization, or reverse transcribed to cDNA. The RT yield, however, varies up to 200-fold on the choice of RT, priming strategy, and mRNA target [15]. As long as the same protocol is used and relative gene expression is compared results are reliable, but the comparison of data from two labs that use different protocols may be tricky. The dominant technique to quantify cDNA is real-time PCR [16]; although, if heating must be avoided, helicase-dependent amplification from New England Biolabs [17] may be an option. Many reporter technologies are available for real-time PCR. SYBR Green [18] and the BEBO [14] dyes are available as non-specific reporters. Since the design of the TaqMan probe [19], a number of other sequence-specific reporter systems have been developed, many of which do not interfere with the PCR reactions, resulting in higher efficiencies. These include AllGlo from Allelogic Biosciences [20], QZyme from Becton Dickinson [21], LNA primers from Exiqon [22], LightUp probes from LightUp Technologies [23], Hyb probes from Roche [24], Molecular Beacons as developed by Tyagi and Kramer [25], Scorpion® primers from DxS [26], LUX™ primers from Invitrogen [27], and the Primer-Probes from WaferGen [3]. The development of quenchers from companies, such as Biosearch Technologies [28], has widened the spectral window for multiplexing using these probes. The main problem in real-rime PCR is the formation of primer-dimer products, which limits the sensitivity of the assays. Primer-dimer products are formed mainly during the preparation of an assay and can be suppressed using Taq polymerase that is inactive until the PCR reaction is initiated. These hot-start systems can be based on chemical modifications of the Taq polymerase, antibody blends (such as presented here by Becton Dickinson [20]), and the new approach based on reversible competition with a synthetic polymer developed by Eppendorf [29]. Hot-start techniques are particularly powerful in combination with probe techniques, where the probing function is an integral part of the primers, or where the probe has an unnatural backbone and can neither prime nor be a substrate for priming. These systems contain fewer oligonucleotides and form less primer-dimer products. Furthermore, better buffer systems, such as Elixir, suppress primer-dimer formation. These developments are important for multiplexing, where primer-dimer formation is harder to suppress because of the larger number of primers and also the total amount of primers that must be used. Becton Dickinson [20], Bio-Rad [30] and the Wadsworth Center reported excellent quadruplex real-time PCR assay data at this meeting. High-throughput quantitarive gene expression analysis is becoming increasingly important in all stages of drug development, vaccine development, plant breeding, and in biodefense research. Advances in sample enrichment, sample preparation, and pre-amplification are important steps toward higher throughput. 2004

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