Método de evaluación utilizando el método de MARKOV para calcular la

Figure 1.15: The process o f classic DDRT-PCR. In some o f the improved techniques adapters at the 5 ' end o f the oligo(dT) and random primers result in more specific reamplification o f the selected bands.

stringency for both the first and second strand cDNA synthesis. PCR is then used to amplify the products (Welsh et aL, 1992). DDRT-PCR is a technique that converts a subset of the mRNA present in the particular tissue or cell into short cDNA fragments using an anchored poly-(A) primer and an arbitrary 10-mer primer. A low annealing temperature (40°C) is required to achieve arbitrary priming. A simultaneous comparison of the cDNAs that are taken from various tissue samples can be made by running the samples side by side on a nondenaturing

polyacrylamide gel, then individual cDNAs specific to a particular cell type or tissue can be isolated (figure 1.14). The technique can be used to identify genes that are active at different stages of development or in different tissues. In addition, it may be useful to detect genes (activated) in response to pharmaceutical drugs or in abnormal tissues such as tumours in comparison to the normal tissue. DDRT-PCR has been cited extensively in the literature and has resulted in several hundred publications reporting its successful application. However, there are many more uncontrolled problems with DDRT-PCR technique than were apparent from the original publications (Debouck, 1995). For instance, false positives, the main disadvantage of DDRT-PCR, comprise a significant portion of isolated fragments. The proportion of false positives (not actually corresponding to DETs) often

reaches 50% or more (Debouck, 1995; Wan et aL, 1996). The most obvious cause of false positives is arbitrarily primed PCR with short (10-12 bases) oligos, in a low stringency PCR,

Therefore, a number of improvements were proposed to overcome its difficulties and fulfil the aims of the DDRT-PCR method and several promising techniques for displaying cDNA fragments were recently developed that differ from the original DDRT-PCR method (Matz and Lukyanov, 1998)(practical details of this method have been discussed in chapter 3). The original method, based on arbitrary primed PCR is usually termed classic DDRT-PCR and the techniques that involve DDRT-PCR combined with more complex methods, such as ODD, GEF and RLCS are named systematic differential display (DD) (Matz and Lukyanov, 1998). The scheme of systematic DD methods basically includes two stages: first, obtaining a sample of cDNA fragments in which each mRNA is represented by a

single fragment of characteristic length; and second, generating simplified non­ overlapping subsets of these fragments consisting of -70-100 members, short enough to be analyzed on a polyacrylamide gel. In this method, the same principle of fingerprint production as ODD, GEF, RLCS and Kato’s method is used: a pool of restriction fragments ligated to adapters is amplified with adapter-specific primers extended by several bases.

4.1.7 Differential display computational tools

Recently specialised informatics tools have been provided to simplify the analysis of the huge accumulative complex data sets which is presented in

databases. Some of them for gene expression analysis such as Digital Differential Display (DDD), cDNA Expression Profiler and Serial Analysis of Gene Expression Map (SAGE) are available at the site of NCBI

(http://www.ncbi.nlm.nih.gov/CGAP/hTGI/). For instance. Digital Differential Display (DDD)(http://www.ncbi.nlm.nih.gov/CGAP/info/ddd.cgi) is a computer method for comparing many fingerprints using results of sequenced ESTs from specific cell, tissue or organ cDNA libraries. Using a statistical test, genes whose expression levels differ significantly from one tissue to the next can be identified and shown to the user.

4.2 Which method is better for displaying of DETs?

The advantage and disadvantage of each technique (the original technique not the improved methods) are shown in table 1.5. Subsequently techniques have been improved to eliminate as many disadvantages as possible until the revised technique sometimes becomes completely different from the original method. The techniques used in a particular situation relate to their advantages and disadvantages. For example, those techniques that are based on PCR amplification are very sensitive and a small amount of starting mRNA can be used but false positive and non­ specific results are seen. In contrast, those using a hybridisation strategy need more mRNA and are not sufficiently sensitive to detect low copy transcripts.

Microarray technique is a good method for DD but for genes which are

already known, requires an expensive equipment and was very new at the beginning of this experiment.

At the time of starting this study, there was a lot of debate in the literature on the possible advantages and disadvantages of classical DDRT-PCR. It has also been indicated that classical DDRT-PCR reveals far less difference than is expected in some well-studied biological systems (Bertioli et aL, 1995; Graf et aL, 1997). This may be attributed to under-representation of the minor mRNA fraction to which most DETs belong.

The results in this thesis were produced using Differential Display RT-PCR. Despite all the problems mentioned, classical DDRT-PCR has a significant

advantage over all other DET search methods in that it is the simplest technique for mRNA comparison and it does not require any special reagents, materials or

instruments such as those needed for array technology. Modified DDRT-PCR techniques which in some publications are called systematic DDRT-PCRs are interesting and sometimes worth using. However, some of those protocols are too complicated to be used widely in the present form and require further development. Classical DDRT-PCR is a satisfactory method, at least in cases when a pronounced difference is expected and there is no need to detect all DETs, for example when searching for tissue markers in cancer tissues or, as is the case of this thesis, searching for novel transcripts unique to a particular cell type.

98

ADVANTAGE AND DISADVANTAGE

(D)

DDRT- PCR

ARRAYS RDA SUBTRACTIVE

HYBRIDISATION LIBRARY SCREENING Advantages Identification of new genes * * * *

Starting from total RNA * *

To obtain full length cDNA and coding

information

*

Comparison of many different samples at a time

*

To obtain 3' end information

* *

Disadvantages

Starting from mRNA * * * *

Hybridisation technology necessary * * * * Time consuming * Expensive equipment necessary *

False positive results * * *

Table 1.5: Advantage and disadvantage o f the main strategies fo r the identification o f DETs.

Clinpter