[Use of the real-time RT-PCR method for investigation of small stable RNA expression level in human epidermoid...

Real-time RT-PCR using fluorescence dyes (e.g. SYBR Green I) is currently the most sensitive and precise method for investigation of RNA level and has long been widely used for absolute and relative quantification of mRNA in the cell. This highly sensitive method allows measurement of different type RNA level in the cell based on the kinetics of the corresponding double-stranded cDNA amplification. Upon its binding to the minor groove of double-stranded DNA, SYBR Green I dye increases its fluorescence about 100-fold, and this increase can be recorded even at early cycles of amplification. During the real-time RT-PCR procedure the level of amplified DNA is measured after every cycle of amplification, which permits to perform quantification at the cycles when amplification curve has not yet reached the "plateau" range and corresponds to the range of exponential increase in DNA amount. This approach makes it possible to avoid misinterpretation of data typical of conventional PCR methods "in the end point" and caused by a deficiency of one or more reaction components at the late PCR cycles. We applied for the first time real-time RT-PCR using SYBR Green I for the measurement of the class III genes RNA-product level, that is, small stable non-translated RNAs--ribosomal 5S rRNA, initiator transfer RNAiMet1, and Alu-RNA, synthesized by DNA-dependent RNA polymerase III. We investigated the level of 5S rRNA-, tRNA- and Alu-gene expression in the cell being in different states: with prolonged generation period, activated to proliferation, and apoptotic. The expression level was judged from the content of corresponding RNA-products in the total cellular RNA. The used approach enabled us to find out the specific RNA share in the total cell RNA. Human epidermoid carcinoma cells A431 were used as a model for investigating class III gene expression level in vivo. These cells expose on their surface an abnormally large amount of receptors to epidermoid growth factor (EGF), and the result of EGF action on A431 cells depends on the growth factor concentration. Low concentrations of EGF (0.1 ng/ml) cause active proliferation of A431 cells, but its high concentrations (10-100 ng/ml) cause apoptosis in these cells. Besides, upon growing in serum-free media, A431 cells continue to proliferate, but by this extending the generation period to 48 h, against 30 h on growing in serum-containing media. Hence, A431 cells can serve as a useful model for investigation of specific gene expression level in cells being in different physiological states, in both slowly and actively proliferating cells, and in apoptotic cells. For successful use of real-time RT-PCR in 5S rRNA, tRNAi(Met)1 and Alu-RNA level quantification, we optimized the amplification reaction conditions. We took into account that the share of each particular RNA in the cell may vary--the share of ribosomal RNA is high, tRNAi(Met)1--low, and Alu-RNA--very low. Moreover, the level of some small RNAs (e.g. Alu-RNA) can vary significantly in cells of different lines. This explains why the amount of cDNA, gained by reverse transcription of total cellular RNA, and the concentration of specific primers used for PCR were different in each case. We showed that the expression of different class III genes--5S rRNA-, tRNA- and Alu-genes, was not similarly regulated in response to external stimuli, causing prolongation of generation period, activation of proliferation and apoptosis. 5S rRNA level was practically the same in A431 cells both having prolonged generation period and being activated by EGF in low concentration, but in apoptotic cells this level dramatically fell about 8-fold. Alu-RNA level was equal in cells with prolonged generation period and in apoptotic cells, and increased about 2-fold in cells activated by EGF in low concentration. The initiator tRNAi(Met)1 level in cells activated by EGF in low concentration and in apoptotic cells was by almost two times higher than in cells with prolonged generation period. The data obtained testify that the real-time RT-PCR method using SYBR Green I yields highly reliable and reproducible quantification for the level of class III gene RNA-products--small stable RNAs (5S rRNA, tRNA and Alu-RNA). Examination of each specific RNA level requires individual selection for the amplification reaction conditions: the amount of cDNA and primer concentration in the sample. This is primarily caused by different expression levels in some particular class III genes within the frames of the cells, and by different levels of some small stable RNAs (e. g. Alu-RNA) in different cell lines. Special attention must be paid to the internal control for discriminating between specific RNA levels in proliferating and apoptotic cells, as in the late apoptosis RNAs of most types are degraded (for example, mRNA of "house-keeping" gene for RPLP0 protein, used as a possible internal control in our experiments). As far as the applied approach allows estimation of a specific RNA share in the total cellular RNA, we propose to chose as internal control mRNA, whose share doesn't change during the total RNA degradation in apoptosis and thus, mRNA degradation is not selective (in relation to other type RNAs). In that way, the real-time RT-PCR method, which is currently the most sensitive and precise method for quantification of RNA in the cell, holds much promise for the investigation of not only different mRNAs, but also small stable RNAs, synthesized by RNA polymerase III.