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 . For matrices of low complexity, the cell-to-signal system from Ambion  lyses cells and is compatible with both RT and PCR. Still another approach is to use filters, such as those developed by Whatman , to purify and store nucleic acids. Frequently, the collected material must be amplified for analysis. For genome analysis, WGA, as developed by molecular staging , 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  and Parallel , or massive parallel sequencing, as developed by 454 Life Sciences . If the DNA is damaged, an option may be to use the DNA polymerase repair enzyme blend called Restorase from Sigma-Aldrich . For direct parallel analysis of RNA, Genentech's  NACA can be used. Alternatively, RNA can be amplified using T7 amplification, or a more advanced variant, such as Ovation from NuGEN , 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 . 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 ; although, if heating must be avoided, helicase-dependent amplification from New England Biolabs  may be an option. Many reporter technologies are available for real-time PCR. SYBR Green  and the BEBO  dyes are available as non-specific reporters. Since the design of the TaqMan probe , 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 , QZyme from Becton Dickinson , LNA primers from Exiqon , LightUp probes from LightUp Technologies , Hyb probes from Roche , Molecular Beacons as developed by Tyagi and Kramer , Scorpion® primers from DxS , LUX™ primers from Invitrogen , and the Primer-Probes from WaferGen . The development of quenchers from companies, such as Biosearch Technologies , 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 ), and the new approach based on reversible competition with a synthetic polymer developed by Eppendorf . 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 , Bio-Rad  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
[Show abstract][Hide abstract] ABSTRACT: Advances in the biologic sciences and technology are providing molecular targets for diagnosis and treatment of cancer. Lymphoma is a group of cancers with diverse clinical courses. Gene profiling opens new possibilities to classify the disease into subtypes and guide a differentiated treatment. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. For accurate gene expression profiling by real-time PCR, several parameters must be considered and carefully validated. These include the use of reference genes and compensation for PCR inhibition in data normalization. Quantification by real-time PCR may be performed as either absolute measurements using an external standard, or as relative measurements, comparing the expression of a reporter gene with that of a presumed constantly expressed reference gene. Sometimes it is possible to compare expression of reporter genes only, which improves the accuracy of prediction. The amount of biologic material required for real-time PCR analysis is much lower than that required for analysis by traditional methods due to the very high sensitivity of PCR. Fine-needle aspirates and even single cells contain enough material for accurate real-time PCR analysis.
[Show abstract][Hide abstract] ABSTRACT: The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.
Molecular Aspects of Medicine 04/2006; 27(2-3):95-125. DOI:10.1016/j.mam.2005.12.007 · 10.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assess-ment of viral load at a given time point, quantitative PCR tests offer the possibility of deter-mining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infec-tion. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR tech-nology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and qual-ity control issues related to standardization and validation of diagnostic assays are discussed.
Molecular Aspects of Medicine 04/2006; 27(2):254-298. DOI:10.1016/j.mam.2005.12.001 · 10.24 Impact Factor
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