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首页> 外文期刊>Journal of Neuroscience Methods >Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining.
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Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining.

机译：使用简单的RT-PCR和溴化乙锭染色对大鼠脑中相对mRNA表达进行定量。

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摘要

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1) subunit (GABA(A)Rbeta(1)) and phospholipase C beta(4) subtype (PLCbeta(4)) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCbeta(4) was significantly higher in cerebellum than cerebrum, and that of GABA(A)Rbeta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.
机译：我们开发了一种使用逆转录聚合酶链反应（RT-PCR）定量相对基因表达的方案，而无需使用放射性同位素，专用设备或额外的核苷酸片段，例如竞争对手。通过比较比率确定大鼠大脑和小脑中GABA（A）受体beta（1）亚基（GABA（A）Rbeta（1））和磷脂酶C beta（4）亚型（PLCbeta（4））的相对基因表达线性扩增靶cDNA片段和3-磷酸甘油醛脱氢酶（GAPDH）cDNA片段产生的PCR产物作为参考。从溴化乙锭染色的琼脂糖凝胶照片的数字化图像中测量PCR产物的密度。 PCR扩增的线性区域在检测系统的线性范围内（单条带中0.3到12 ng DNA）。本方法的准确度是单次测定中基因表达的差异小于2倍，重复测量后差异为1.5倍，具有统计学意义。估计的PLCbeta（4）相对表达在小脑中明显高于大脑，而GABA（A）Rbeta（1）在这两个区域中相同。使用本方法，可以量化已知离子通道，神经递质受体和细胞内信号传导酶基因家族的几种不同的亚基和亚型。

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