The mutual adjustment of the expression of various genes within a cell a fundamental control process at the work within all living bodies, which is to say that t her e is a gene expr ession network. To analyze this kind of adjustment mechanism, the amount of mRNA, which is a transcript product or the amount of protein, which is a translation product can be masured directly by Kronos Dio. However, the reporter gene assay using luciferin-luciferase reaction is widely used because the expression can be quantitatively monitored more easily and more sensitively.
GFP and firefly luciferase are used as a reporter protein. GFP is fluorescent protein and it is widely used as a reporter of the gene expression. The search results of PubMed in 2005 are shown in the table above. One characteristic point is that in comparing total number of hit, the number of GFP is larger but, luminescent protein is more frequently used for studies related to transcription factors. GFP is an extremely stable protein that absorbs light of 470 to 490 nm and emits green fluorescence. It does not require a luminescent substrate such as luciferase, so it is advantageous in terms of cost, and it is a very convenient reporter when confirming the presence or localization of expression over time with Kronos Dio.
Luciferin shows specific luminescent reactions with luciferase and has few nonspecific reactions, therefore its S/N is satisfactory and it has excellent quantitativeness. It is also considered that cell toxicity from luciferin is small, so that it is possible to monitor fluctuation of transcriptional activity in real-time for extend periods by just resolving luciferin in the medium. However, when measuring fluctuations of expression over time, the fact that GFP is a stable protein becomes a disadvantage and it becomes difficult to monitor subtle difference. (It has become possible to obtain GFP with a shorter life in order to compensate for this disadvantage.) Moreover, for measuring the expression amount of the gene over time, it is necessary to assume that excitation light irradiation may easily damage the cell. Firefly luciferase is a protein with a molecular-size of approx. 62kDa, and it is normally considered as having a shorter half-life of around 3 hours. Luciferase with an even shorter life can also be used. Luciferin, the luminescent substrate is sold as an item enclosed in a kit together with a crystal product, cytolysis agent and reactive reagent, so it is utilized according to different needs.
As mentioned at the beginning, the method of measuring the amount of mRNA is also used normally. When this method is used, a cell needs to be crushed to extract RNA, so when measuring temporal changes, it is necessary to prepare cells of the same quality for the amount equivalent to the number of measurement times. Besides, RNA needs to be extracted within a limited time, so it becomes quite a serious task. It is also known that there is not necessarily a correlation between the amount of RNA and the amount of the translation when comparing the amounts of the two kinds of RNA.