Chen C K, Kinsman S L, Holtzman D M, Mobley W C, Johnston M V
Kennedy Krieger Research Institute, Johns Hopkins University School of Medicine, Baltimore 21205.
Int J Dev Neurosci. 1994 Jun;12(4):255-62. doi: 10.1016/0736-5748(94)90072-8.
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)
神经营养因子的作用是通过特定受体介导的。神经生长因子（NGF）作为典型的神经营养因子，可与高亲和力和低亲和力的受体结合。一种大小为75 kDa的蛋白质（p75NGFR）以低亲和力结合NGF，以及脑源性神经营养因子和神经营养因子3。最近的研究表明，这种蛋白质可能也是高亲和力NGF受体复合物的一个组成部分。为了研究p75NGFR分子的基因表达，我们使用了一种灵敏的逆转录-聚合酶链反应（RT-PCR）测定法来测量其信使核糖核酸（mRNA）在总RNA小样本中的水平。该测定法基于使用缩短的p75NGFR互补核糖核酸（cRNA）作为内部RNA标准，以控制逆转录和聚合酶链扩增中的变异性。我们测量了大鼠小脑在个体发育过程中p75NGFR mRNA的水平，以进一步研究已知在出生后早期该脑区发生的受体基因表达的短暂发育性增加。我们发现，p75NGFR mRNA水平在出生后第2天最为丰富，然后在整个出生后发育过程及成年期下降至较低水平。对我们RT-PCR测定中使用的相同总RNA样本进行的Northern印迹分析证实，p75NGFR表达在出生后早期最高。这些结果证实了先前使用核糖核酸酶保护或Northern印迹测定法、用大量RNA完成的研究结果。使用带有内部标准的RT-PCR测定法还可控制RNA复杂性的变化，而这种变化会影响跨发育阶段的信使丰度测量。（摘要截短于250词）
