Semiquantitative polymerase chain reaction in RNase-producing tissues: Analysis of the developing pancreas.
OBJECTIVE
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.
Hembree
M J
MJ
Children's Mercy Hospital, Director, Laboratory for Surgical Organogenesis, Children's Mercy Hospital, 2401 Gillham Rd, HHC 623C, Kansas City, MO 64108, USA.
Prasadan
K
K
Manna
P
P
Preuett
B
B
Spilde
T
T
Bhatia
A
A
Kobayashi
H
H
Buckingham
B
B
Snyder
C L
CL
Gittes
G K
GK
D000818:Animals; D002452:Cell Count; D002455:Cell Division; D018507:Gene Expression Regulation, Developmental; D005865:Gestational Age; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D051379:Mice; D010179:Pancreas / Q000166:cytology / Q000196:embryology / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000032:analysis*; D014404:Tubulin / Q000032:analysis* / Q000378:metabolism
D007328:Insulin; D012333:RNA, Messenger; D014404:Tubulin; D012260:Ribonucleases