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Updated: Jul 20, 2026
RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue
Published on: August 2, 2014
Semiquantitative polymerase chain reaction in RNase-producing tissues: Analysis of the developing pancreas
M J Hembree1, K Prasadan, P Manna
1Children's Mercy Hospital, Director, Laboratory for Surgical Organogenesis, Children's Mercy Hospital, 2401 Gillham Rd, HHC 623C, Kansas City, MO 64108, USA.
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.
