| First Author | Hembree MJ | Year | 2001 |
| Journal | J Pediatr Surg | Volume | 36 |
| Issue | 11 | Pages | 1629-32 |
| PubMed ID | 11685688 | Mgi Jnum | J:74177 |
| Mgi Id | MGI:2157707 | Doi | 10.1053/jpsu.2001.27934 |
| Citation | Hembree MJ, et al. (2001) Semiquantitative polymerase chain reaction in RNase-producing tissues: Analysis of the developing pancreas. J Pediatr Surg 36(11):1629-32 |
| abstractText | BACKGROUND/PURPOSE: Many studies in pediatric surgical research use a quantitative analysis of gene expression in microscopic quantities of tissue. The authors describe an analysis of the beta-tubulin mRNA content of the embryonic pancreas, which contains abundant endogenous RNases. A detailed analysis of this RNase-containing system will provide a good template for analysis of other potentially simpler systems. METHODS: Embryonic mouse pancreases were harvested at serial gestational ages. DAPI nuclear staining allowed for counting of cells. cDNA was amplified using a fluoresceinated primer and the normalized fluorescence determined. Known numbers of molecules were amplified in parallel as a standard control. RESULTS: The number of cells increased from 38,000 to 2,700,000 between embryonic day 10.5 (E10.5) and E18.5. mRNA for beta-tubulin did not increase proportionately. Assuming a yield of 100% at E10.5 when no RNases are present, the yield of expected mRNA was 65.3% at E12.5, 13.8% at E15.5, and 0.9% at E18.5, presumably because of the appearance of RNases. CONCLUSIONS: Several parameters must be considered in performing semiquantitative reverse transcription polymerase chain reaction: (1) the yield of RNA based on the projected amount of mRNA, (2) the number of cells in the tissue, and (3) a known number of template molecules amplified in parallel. |
