A reverse transcription-polymerase chain reaction study of p75 nerve growth factor receptor gene expression in developing rat cerebellum.
The actions of the neurotrophins are mediated through specific receptors. Nerve growth factor (NGF), the prototypic neurotrophin, binds to receptors of both high and low affinity. A protein 75 kDa in size (p75NGFR) binds NGF, as well as brain-derived neurotrophic factor and neurotrophin 3, with low affinity. Recent investigations suggest that this protein may also be a component of the high affinity NGF receptor complex. To study gene expression of the p75NGFR molecule, we used a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure levels of its messenger RNA (mRNA) in small samples of total RNA. The assay is based on using a shortened p75NGFR cRNA as an internal RNA standard to control for variability in reverse transcription and polymerase chain amplification. We measured p75NGFR mRNA levels in the rat cerebellum during ontogeny to further study the transient developmental increase in receptor gene expression known to occur in this brain region during the early postnatal period. We found that p75NGFR mRNA levels were most abundant at postnatal day 2, and then declined to lower levels throughout postnatal development and in the adult. Northern blot analysis of the same total RNA samples used in our RT-PCR assay verified that p75NGFR expression is highest in the early postnatal period. These results confirm those of previous studies accomplished with much larger amounts of RNA using ribonuclease protection or northern blot assays. The use of an RT-PCR assay that utilized an internal standard also controls against changes in RNA complexity which can affect the measurement of message abundance across developmental stages.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
C K
CK
Kennedy Krieger Research Institute, Johns Hopkins University School of Medicine, Baltimore 21205.
Kinsman
S L
SL
Holtzman
D M
DM
Mobley
W C
WC
Johnston
M V
MV
D000375:Aging / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002531:Cerebellum / Q000254:growth & development* / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017475:Receptors, Nerve Growth Factor / Q000235:genetics*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D017475:Receptors, Nerve Growth Factor