11. Conclusion

tags for sequencing. LongSAGE enables transcriptome analysis of increased lengths which in

Microarrays provide a global view of gene expression and are found in two forms; DNAfragment-based and oligonucleotide-based microarrays [65] with the source of array fragments being either anonymous genomic clones, EST clones or ORF amplified DNA fragments. The advantage of this technique is that a range of both weak and strong signals can be monitored on the same microarray, enabling the simultaneous analysis of a large number of genes. In addition, the technique allows for a pair-wise comparison of samples [66]. However, a major disadvantage of this technique is that an accurate sequence database must be available to facilitate the construction of the microarrays, as well as a large amount of mRNA as starting

With the advent of next-generation sequencing, RNA sequencing (RNASeq or whole transcriptome shotgun sequencing), was developed. RNA-Seq is used to analyze changes in the different RNA species comprising the cellular transcriptome and can inform on the presence and quantity of RNAs in plant samples [67]. Specifically, RNA-Seq facilitates the ability to look at genetic alterations, mutations and changes in gene expression, or differences in gene expression in different groups or treatments such as transgenic – vs. conventional plants.

However, all of the above techniques require substantial amount of sequence information of the genome under investigation. Moreover, availability of funding is another factor for consideration. As a result, alternative gene expression techniques can also be investigated for suitability of intended use [61]. These include mRNA Differential Display (DD), Representational Difference Analysis (RDA), Amplified Fragment Length Polymorphism (AFLP) and quantita-

Differential gene expression analysis was first performed using mRNA Differential Display (DD) [68]. During DD, cDNA is synthesized from mRNA of each sample of interest, followed by amplification using a combination of anchored oligo-dT and random oligonucleotides. The obtained differentially amplified fragments each represent a transcript or an expressed sequence tag (EST). The advantage of this technique is that it requires a small amount of bioinformatics application during data analysis. Improvements of the technique generated the second generation annealing control primer (ACP)-differential display RT-PCR [69]. Under optimal conditions of use, mRNA DD is a relatively inexpensive but powerful tool, used to identify and isolate differentially expressed transcripts, as well as for comparative studies

Subtractive hybridization of mRNA is another method that has been employed to differentially identify mRNAs associated with a cell- or tissue type or cellular responses [71]. A reduction in the number of genes in need of analysis in a comparative transgenic study is an important advantage [72]. Another advantage of the technique is its ability to reveal lower abundance transcripts [73], but the technique is also time consuming and labor intensive.

Representational Difference Analysis (RDA) is a subtractive DNA enrichment technique designed to identify differences between two genomes without quantifying expression levels [74]. The technique was later modified by using cDNA as template to facilitate the detection of rare transcripts. cDNA-RDA utilizes subtractive DNA enrichment in association with PCR

turn improves the accuracy of annotating genes [64].

124 Transgenic Crops - Emerging Trends and Future Perspectives

material to perform the gene expression analysis [65].

tive reverse transcriptase real time PCR (qRT-PCR).

between several mRNA populations [70].

Due to the non-selective nature of traditional methods of genetic modification, the possibility exists that endogenous genes and their functions will be disrupted through the random integration of the transgene into the plant genome. This phenomenon is linked to unintended effects of genetic modification. Gene expression analysis is thus a crucial part of investigations into the effect of transgene insertion on endogenous gene expression. An understanding of the dynamics of the various available techniques is thus important in selecting the most appropriate technique(s) for the realization of the set objectives. Each method described above has its advantages and limitations. Furthermore, the choice of technique would depend on whether prior knowledge of the host genome is available or not. Using more than one technique in complement would ensure optimum results for investigating comparative / differential gene expression analysis in transgenic crops.
