Relative quantitative RT-PCR protocol for TrkB expression in neuroblastoma using GAPD as an internal control

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.