Methods of gene expression analysis

Methods of gene expression analysis

From 2001, when the encoded genome of the human being was announced, many science and industry forces were turned to the investigations of gene expression. Even now discussions still take place between the advocates of mRNA expression analysis and protein analysis, which method more realistically reflects the functional gene expression. mRNA methodology is characterised by fastness, completeness and sensitivity, nevertheless, sometimes a difference between the whole of organism genes and the phenotype thereof, for example, that a butterfly and its caterpillar have identical genomes, but completely different proteins. But the fact is that both mRNA and protein expression are techniques supplementing each other, each of which provided an irrevocable contribution into the investigation of gene expression, and which technique will be chosen by the investigator depends only upon the goals set by him and the financial resources.

Before going deeper into separate methods of investigation a general notion of the gene expression should be possessed.

The bases of transcription and transmittance taking place within the cell

Proteins are very important in almost all biological processes. They perform the catalysis of chemical reactions, determine the stability and structure of the cell control the entrance of the molecules important to the cell and protection and are responsible for the growth end evolution of the cell. The structure of the proteins is determined by the sequence of DNA bases, but this information is not transmitted directly into the proteins (Figure 1). During the transcription, mRNA molecule copies the information of the DNA and one chain of mRNA transmits the information into ribosomes and there mRNA molecule is “turned into” the chains of amino acids of the respectful sequence. It encodes the information, what amino acids from the 20 possible ones and in what order will be synthesised.


Figure 1. Scheme of movement of genetic information within the cell. Genetic code, which is preserved in DNA sequences, is the “plan”, upon which the synthesis of the protein will depend. Nevertheless, the information from DNA is not transmitted directly into the proteins, but first of all, during transcription it is transmitted into pre-RNA. It is identical to the encoding DNA sequence, the only difference is that thymine is replaced by uracil. During the splicing process, not encoding parts of RNA (introns) are eliminated and in this way mRNA is received. Both ends of mRNA are modified. Most often the end 5′ has a cap, while the end 3′ has a tail (poli(A)). mRNA from the nucleus goes to cytoplasm and there the synthesis of proteins takes place within the ribosomes.

Methods of gene expression analysis

In 1990 application of different gene expression technologies as started, looking for answers into various biology questions. Nevertheless, from then on the attitude of scientists towards the analysis of gene expression changes significantly: at that time possibilities did not allow analyzing one gene using Northern or Western blotting method, while now it is possible at the same time to evaluate the expression of almost all genes of the genome. Analysis of gene expression became one of the most widely used techniques in biomedical researches.


Figure 2. Classification of methods of gene expression analysis

Methods of functional gene expression analysis are subdivided into 2 main large groups: those of RNA expression and protein expression. These groups, depending on the possibilities of the methods, can be subdivided into subgroups, i.e. systems of “closed architecture” and “open architecture”. It is also possible to subdivide gene expression analysis methods based on RNA according to the procedures applied for their performance. These methods are subdivided into 3 groups: based on hybridization, based on PCR reaction and based on sequencing. Typical example of a method based on hybridization is DNA micro-camera method, example of method based on PCR – PCR method based on inverted transcription (AT-PCR) and example of method based on sequencing – method of serial gene expression analysis (SAGE). But in fact none of the methods is used in the investigative work, for example, in investigations of differentiated transcript expressions amplified expression of differentiated genes (ADGE) is used in combination with technique based on PCR and DNA micro-camera technique.

Closed architecture systems, such as DNA micro-cameras, allow analyzing known genes, while open systems, such as differentiation method based on PCR, allow analyzing genes, which were previously unknown. Qualitative analysis of genome transcriptomes, using closed system, depends directly upon the possessed information about the genome. DNA micro-cameras and qualitative AT-PCR (inverted transcription) methods, for example, of competitive PCR and real-time PCR, are the methods which are most often used in gene expression investigations. Application of closed systems is still restricted. Even in those species, the genome of which is known, transcriptomes proved to be more complicated than it was possible to foresee according to the number of genes, because they were influenced by splicing, variety of genes, RNA amendment and etc.

Otherwise than the closed architecture systems, open architecture systems do not require the initial information on the transcriptomes. At least in theory, using open architecture system it is possible to identify all RNA which were created during splicing, repaired or modified in any other way, because that has no influence upon the quality of expression analysis. Nevertheless, this system can not widely and in a versatile way comprise the whole transcriptome.

mRNA expression methods


Figure 3. Scheme of mDNA cloning based on PCR.