Gene-expression analysis of single cells-nested polymerase chain reaction after laser microdissection

Figure 1 Examples of laser microdissection in colon tissues (hematoxylin and eosin, magnification × 200). Single cells were picked by combination laser-manipulated microdissection (LMM) and laser pressure catapulting (LPC). A, C, E, G: Before Laser microdissection. B, D, F, H: Using LMM, the laser precisely circumcised selected cells, yielding a cut gap between selected and non-selected areas. Then the selected cells were catapulted using the LPC technique. A-F: Typical images before and after LMM and LPC. G, H: Laser microdissection could also be used to cut out the nucleus of a selected cell.

Figure 2 Amplification results from nested PCR of colon cells after laser microdissection. Nested PCR amplification products of DNA of single cells could clearly be visualized by agarose gel electrophoresis. A: Amplification results of β-actin-outer primers. B: Amplification results of β-actin-inner primers. M: DNA markers (upper to lower: 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp). 1: PCR positive control; 2: one complete section; 4, 6 and 8 are 100, 10 and 1 cell(s), respectively; 3, 5, 7 and 9 are negative controls of 2, 4, 6 and 8; 10: PCR negative control.

Figure 3 Amplification results from nested RT-PCR for cDNA of single cells after laser microdissection. A: Amplification results of β-actin-outer primers. B: Amplification results of β-actin-inner primers. M: DNA markers (upper to lower: 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp). 1: PCR positive control; 2: one complete section; 4, 6, 8 and 10 are 200, 100, 10 and 1 cell(s), respectively; 3, 5, 7, 9 and 11 are negative controls of 2, 4, 6, 8 and 10; 12: PCR negative control.