Detection of mRNA Expression and Alternative Splicing in a Single Cell

We have developed a method to detect mRNA expression in a single cell without isolating RNA from the cell (1 ). The method includes the following manipulations:detach cells, pick a single cell, destroy the cell by heating, reverse-transcribe RNA, and PCR-amplify. Using this method, we succeeded in detecting the expression of an abundant mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This is a simple and easy method to detect the expression of abundant mRNAs. However, the method failed to detect fibronectin (FN)mRNA in fibroblasts—which express significant amounts of FN—probably because the expression level of FN mRNA was not adequate for the single-step method. Next, we developed another method (2 ). This new method is composed of two steps, a variation of nested PCR. Like the old method, the first step includes the following manipulations:detach cells, pick a single cell, destroy the cell by freezing and thawing, reverse-transcribe RNA, and PCR-amplify. The second step is composed of PCR amplification with another nested PCR-primer set. Using this nested PCR technique, we could detect the expression of FN. We also succeeded in analyzing alternative splicing of FN mRNA in a single cell.