Measurement of interleukin-1 stimulated constitutive prostaglandin G/H synthase (cyclooxygenase) mRNA levels in osteoblastic MC3T3-E1 cells using competitive reverse transcriptase polymerase chain reaction.
To assess regulation of constitutive prostaglandin G/H synthase (PGHS-1) by interleukin-1 (IL-1) in osteoblastic MC3T3-E1 cells, we compared analysis by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with Northern blot analysis. Using RT-PCR, IL-1 increased PGHS-1 mRNA levels by 1.84 +/- 0.10 or 2.07 +/- 0.17, depending on the method of calculation. Using Northern blot analysis, the effect of IL-1 on PGHS-1 mRNA levels was more variable, and the variability was increased by normalization of PGHS-1 mRNA levels to the housekeeping genes, beta-actin and glyceraldehyde phosphate dehydrogenase (GAPDH), because their mRNA levels were also regulated by IL-1. We conclude that competitive RT-PCR is a reproducible and accurate method for studying small changes in mRNA levels.
Kawaguchi
H
H
Department of Medicine, University of Connecticut Health Center, Farmington 06030.
Yavari
R
R
Stover
M L
ML
Rowe
D W
DW
Raisz
L G
LG
Pilbeam
C C
CC
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D007375:Interleukin-1 / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010006:Osteoblasts / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase
D007375:Interleukin-1; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011451:Prostaglandin-Endoperoxide Synthases; D012194:RNA-Directed DNA Polymerase