Gene expression profiling methods

Methods for quantifying mRNA abundance in various plant tissues and experimental conditions are: (i) RNA gel-blot (northern) analysis, (ii) differential display (24), (iii) quantitative real-time PCR, (iv) cDNA-amplified fragment length polymorphism (AFLP) analysis (25), (v) serial analysis of gene expression (SAGE) (26), (vi) massive parallel signature sequencing (MPSS) (27), (vii) cDNA macroarray analysis (28), (viii) cDNA microarray analysis, and (ix) oligonucleotide microarray analysis. These methods have several advantages and disadvantages (7, 8, 29).

RNA gel-blot (northern) analysis is an established and reliable method, which allows accurate quantification of specific transcripts, but it cannot be applied for genome-scale expression analysis.

Differential display uses low stringency PCR, a combinatorial primer set, and gel electrophoresis to amplify and visualize larger populations of cDNAs representing mRNA populations of interest. Differential display is a relatively cheap and simple means of screening for differentially expressed genes, and is particularly useful when the availability of RNA is limited. However, this technique requires a large number of reactions to achieve maximal coverage of all active transcripts and suffers from an output that is not quantitative and identified sequences are often difficult to clone and confirm (8).

Quantitative real-time PCR (QRT-PCR) has been demonstrated to generate robust, quantitative expression data for single genes and this method offers rapid and reproducible results (30). One of the major advantages of real-time PCR is its broad dynamic range with which one can precisely quantify transcript concentrations over more than at least five orders of magnitude (31). RNA gel-blot analysis and real-time PCR are often used to confirm differential expression of genes detected by DNA microarray analysis.

The principles of AFLP are applied to cDNA templates in cDNA-AFLP analysis, which has been used to identify differentially expressed genes involved in a variety of plant processes. This technique offers several advantages over traditional approaches. Of particular importance is the fact that poorly characterized genomes can be investigated in a high-throughput manner. Because the stringency of cDNA-AFLP PCR reactions is quite high (which is not the case with differential display) the fidelity of the cDNA-AFLP system allows much greater confidence in acquired data and differences in the intensities of amplified products can be informative (25). As with the other profiling methods described here, the sensitivity of cDNA-AFLP is only limited by the ability of cDNA libraries to capture low-abundance transcripts (7).

SAGE is based on the capture and sequence analysis of a short region close to the 3' end of each cDNA in the sample (26), and it is a quantitative or digital method of gene expression analysis like EST sequencing. SAGE is time-consuming and requires an extensive foundation of sequence information. Variations in amplification efficiency between ditags may lead to distorted results (29).

MPSS, developed and commercialized by Lynx Therapeutics (Hayward, CA), is based on methods to clone individual cDNA molecules on microbeads and sequence, in parallel, short tags or signatures from these cDNAs (27). The final output of MPSS is a set of abundances for thousands of distinct 17- or 20-base signatures, most of which uniquely identify a particular transcript. The parallel sequencing method produces millions of MPSS signatures in only a few weeks, but the technology is sufficiently complex, and unlike SAGE, it cannot be performed in individual laboratories (8).

cDNA macroarray technology allows parallel and comparative analysis of the expression of thousands of genes (28) as well as DNA microarray technology. The cDNA macroarrays and the cDNA microarrays differ primarily in the type of solid support immobilized: that is, the macroarrays use a membrane-based matrix while the microarrays use a glass or plastic slide. In most cases, macroarray targets are radioactively labeled. The cDNA

macroarray analysis is also less expensive than the oligonucleotide array analysis. However, it takes a lot of work, such as printing on many membranes, to prepare a whole genome macroarray. A number of reviews have described how cDNA macroarrays are used and the advantages and disadvantages of cDNA macroarray analysis (32, 33).

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