Deep sequencing – a new method and new requirements of gene expression analysis
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The determination of gene expression is a very common scientific method used in modern laboratory for a variety of applications. One of the most popular is the real time PCR, a quantitative modification of the classic PCR method where the increase of the amplify nucleic acid is examined cycle by cycle after every amplification step. The analysis of the PCR productduring the amplification process allows to compare the initial amount of cDNA synthesized from isolated RNA and calculate the number of particular RNA copies present in examined material. In spite of obvious advantages of real time PCR there are also some inconveniences of this method. First of all, there is no possibility of analyzing more than one gene in a single reaction mixture. It is limited by the necessity of design and usage of different pairs of primers for each analyzed gene. Therefore, it is necessary to predict the cell, tissue or organism response for applied treatment, examined condition, etc. The development of microarray methods enables to overcome these problems and parallel analyze all known genes in the single sample at the same time. There is no need to predict which gene expression might be changed under studied conditions because the microarray data is a comprehensive pattern of the expression of all known genes, which probes are implemented on the microchip surface. Although the microarray data is an excellent method for gene expression comparison, the estimation of the extent of change fold is not very precise and usually is confirmed and determined by real time PCR with respect to selected genes. The method which combines the quantitative precision of real time PCR and the possibility to analyze broad spectrum of the genes is a deep sequencing method also called next generation sequencing. It is a new method developed for the analysis of the whole RNA isolated from a sample without the need to design primers and thus any knowledge of expressed genes sequence. The advantages of this method include the possibility of finding unexpected expression of completely unknown DNA fragments, alternative splicing variants of the genes and differences in DNA sequence. The deep sequencing provides an extremely large amount of information, much more than microarray data, and to analyse it new bioinformatics methods and tools especially designed for this purpose are required.
- Department of Biology and Pathology of Human Reproduction, Institute of Animal Reproduction and Food Research of Polish, Academy of Sciences in Olsztyn
- Department of Cytobiochemistry, Institute of Biology, University of Bialystok
- Department of Reproduction and Gynecological Endocrinology Medical University of Bialystok
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