Relative quantitative RT-PCR protocol for TrkB expression in neuroblastoma using GAPD as an internal control
Relative quantitative RT-PCR protocol for TrkB expression in neuroblastoma using GAPD as an internal control
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DOI：
10.2144/00284st04
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发表时间：
2000-04-01
期刊：
影响因子：
2.7
通讯作者：
Ikegaki, N
中科院分区：
文献类型：
--
作者：
Eggert, A;Brodeur, GM;Ikegaki, N
描述了使用甘油醛-3-磷酸脱氢酶（GAPD）转录物作为内部对照用于TrkB转录物的相对定量测量的RT-PCR方案。使用5 '-生物素化引物在扩增的指数期共扩增TrkB和GAPD序列。PCR产物经聚丙烯酰胺凝胶电泳，电转移到尼龙膜上，用碱性磷酸酶偶联抗生物素蛋白进行荧光检测。通过光密度法分析在X射线胶片上检测到的信号。确定TrkB和GAPD表达水平之间的比率以标准化TrkB转录物的表达水平。最初：与TrkB相比，GAPD转录物的强信号导致X射线胶片的过度曝光，这导致难以准确测定TrkB/GAPD比率。为了避免这个问题，用具有相同序列和浓度的生物素化和非生物素化的GAPD引物的混合物替换均匀生物素化的GAPD引物。随着生物素标记引物伊拉总GAPD引物中所占比例的减少，X射线胶片上检测到的GAPD信号成比例地减少。该修饰使得能够在X射线胶片检测的线性范围内分析GAPD和TrkB信号，而不影响TrkB序列的扩增效率。复合引物的使用在核酸定量分析中具有广泛的适用性。
An RT-PCR protocol for the relative quantitative measurement of TrkB transcripts using glyceraldehyde-3-phosphate-dehydrogenase (GAPD) transcripts as an internal control is described Both TrkB and GAPD sequences,were co-amplified in the exponential phase of amplification using 5'-biotinylated primers. The PCR products were subjected to PAGE, electro-transferred to nylon membrane and detected by a chemiluminescent procedure using alkaline phosphatase conjugated,with avidin. Signals detected on X-ray film were analyzed by densitometry. The ratio between TrkB and GAPD expression levels was determined to normalize the expression levels of TrkB transcripts. Initially: strong signals of GAPD transcripts led to overexposure of X-ray film compared to those of TrkB, which causes difficulties in the accurate determination of the TrkB/GAPD ratio. To circumvent this problem, uniformly biotinylated GAPD primers were replaced with a mixture of biotinylated and non-biotinylated GAPD primers of the same sequence and concentration. GAPD signals detected on X-ray film proportionally decreased as the amount of biotin-labeled primers was reduced ira the total GAPD primers. This modification enabled both GAPD and TrkB signals to be analyzed within the linear range of X-ray film detection without affecting the amplification efficiency of TrkB sequence. Use of composite primers,,may have a wide range of applicability in quantitative analysis of nucleic acids.