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 were proportionally decreased as the amount of biotin-labeled primers was reduced in 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.
Eggert
A
A
Children's Hospital of Philadelphia, PA, USA.
Brodeur
G M
GM
Ikegaki
N
N
D019921:Biotinylation; D015152:Blotting, Northern; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D020813:Receptor, trkB / Q000096:biosynthesis* / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D020813:Receptor, trkB