The β-actin mRNA expression of individual living cells was examined using rat fibroblast-like VNOf90 cells and mouse osteoblast-like MC3T3-E1 cells (Fig. ). Although β-actin mRNAs are usually distributed throughout the cytoplasm uniformly, they are localized to the leading edge of the cells when the cells start to migrate [22
]. Thus we chose the single cells surrounded by other cells that inhibit the migration of the target cells. PCR products for rat and mouse β-actin mRNAs were detected as shown in the even numbered lanes of Figs. and . In the negative control, PCR products were not detected without the insertion of the tip into the cell (odd numbered lanes in Figs. and ). Experiments for the detection of β-actin mRNA and the negative control were performed alternately.
Figure 1 Principal features of the experimental procedure. A target region of a cell on a Petri dish was positioned underneath the AFM tip through the observation of an inverted optical microscope combined with AFM (a). The AFM tip was then lowered onto the cell (more ...)
In Table , the detection of β-actin mRNA from single VNOf90 cells is presented. We performed our new method on 102 single living cells. The number of assays against single cells ranged from one to six. The interval time between one assay to the next one against the same cell ranged from 5 to 60 min. When we performed assays six times against three single cells, the following results were obtained. In cases of two single cells, PCR products were detected at all six assays. In case of another cell, PCR products were detected five times out of 6 assays. Seventy-two positive results were obtained when we tried one assay against 73 single cells. In total, we performed 189 assays on 102 single living cells, and 182 positive results were obtained (96.3%). We encountered seven negative results which may have resulted from the following three reasons:
The detection of β-actin mRNA from single VNOf90 cells.
1) The procedure for inserting the tip into the cell was not successful.
2) The PCR reaction was not successful.
3) β-actin mRNA did not exist in the cell where the tip was inserted.
The first two results were considered to be experimental errors, and the last one was considered to result from the probability of the existence of mRNA. Nevertheless, the high probability of detecting β-actin mRNA indicated that it could be used as a positive marker for examining the expression of other genes which would exclude the first two reasons from consideration. A population of mammalian cells grown in culture medium contains about one million mRNAs per cell. Fibroblast cells are reported to contain 2,000~3,000 copies of β-actin mRNA per cell [24
]. Our method can be applied to detect other mRNAs whose copies number more than several thousand per cell. To extract mRNA specifically or detect smaller numbers of mRNA, the use of AFM tips coupled with oligo(dT)n might be helpful.
In the next experiment we tried to examine if our method could be used to determine more than one kind of gene at the same time. As shown in Fig. , VNOf90 cells expressed fibronectin protein as well as β-actin. AFM tips inserted into the single living cells were placed into RT-PCR solution that contained primers for both β-actin and fibronectin. Five μl aliquots of the first PCR product were put into a second PCR solution containing β-actin or fibronectin primer sets. In total, we assayed 10 single living cells and the results of five single cells are shown in Fig. . PCR products for both β-actin and fibronectin mRNAs were detected in all of the 10 different single cells.
Figure 2 Detection of both β-actin and fibronectin mRNAs from single living cells. Immunocytochemistry demonstrated VNOf90 cells expressed both fibronectin (a) and β-actin (b). Both β-actin and fibronectin mRNAs were detected in all of (more ...)
In order to examine time dependent gene expression of single living cells, the response of rat VNOF90 cells to serum has been used as a model for studying the changes in c-fos gene expression (Fig. ). In our study the cells were induced to enter a quiescent state with DMEM containing 0% FBS for 24 hours and then stimulated by addition of 10% FBS and 10 μg/ml cycloheximide (CHX) for 2 hours. CHX is well known to induce increases in c-fos mRNA expression level to prevent degradation of c-fos mRNA. The cell medium was then changed to DMEM containing no FBS and 10 μg/ml CHX for one hour. Finally, the cell medium was changed to DMEM containing 10 μg/ml CHX (Condition A) or no CHX (Condition B). Total RNA from confluent cells on 60 mm dishes at different times was extracted using the procedures of the RNA Isolation Kit (Gentra System, MN, USA). Quantification of mRNA was carried out with quantitative PCR. The β-actin mRNA levels were more constant than the c-fos mRNA levels in both A and B conditions (Fig. ). Although the c-fos mRNA levels decreased rapidly in the absence of CHX from 3 to 5 hours following stimulation, they decreased gradually in the presence of CHX from the cell population analysis. The detection of c-fos and β-actin mRNAs from single cells, termed A1, A2, and A3 cells (from condition A) was examined with our method (Fig. ). The change of β-actin mRNA levels was also small compared with that of c-fos mRNA levels in single cell analysis as well as in the cell population analysis. Although the expression profile of c-fos mRNA in the A1 cell was very similar to the result obtained from the cell population analysis, the A2 and A3 cells showed different expression profiles. These results suggest that c-fos mRNA expression levels are not uniform among individual cells. Although c-fos mRNA was not detected in the A3 cell at 3.5 h, β-actin mRNA was detected, indicating that the experimental errors were able to be eliminated. In condition B (B1, B2, and B3 cells), c-fos mRNA expression was not observed in any of the cases at 5 h following stimulation. This result corresponded to the result from the cell population analysis. The expression of c-fos mRNA in the B1 cell was observed only at 3 h. In the B2 cell, c-fos mRNA expression was detected at 3 h and at 3.5 h and not after 4 h. In the B3 cell, c-fos mRNA expression was detected at 3 h and not at 3.5 h, and then detected again at 4 h. This type of pattern was also observed in the A3 cell. These results indicate the possibility that the fluctuation of c-fos mRNA expression level occurs at the single cell level. Figure shows the histogram of the number of β-actin mRNAs bound to AFM tips. In most cases, their number was equal to or less than 20. We performed nested PCR as well as quantitative PCR followed by RT-PCR to examine c-fos mRNA expression. When c-fos mRNA was not detected with quantitative PCR, the nested PCR product was not detected on agarose gels, either.
Figure 3 The cell population analysis. The response of VNOf90 cells to serum and CHX was used as a model for studying time dependent gene expression (a). VNOF90 cells were induced to enter a quiescent state by serum deprivation for 24 hours and then stimulated (more ...)
Figure 4 The single cell analysis. Time dependent mRNA expression of single living cells was examined (a and b). The ordinate represents a common logarithm of initial mRNA copies. The nd on the ordinate indicates that the product was not detected with quantitative (more ...)