COMPARATIVE-ANALYSIS OF MULTIPLE TECHNIQUES FOR SEMIQUANTITATION OF RT-PCR AMPLICONS

Reverse-transcription polymerase chain reaction (RT-PCR) was used to g enerate a panel of distinct amplicons from total RNA derived from a va riety of sources. Following amplification, semi-quantification of the different amplicons was performed by normalization to steady-state tra nscript levels of the house-keeping gene, glyceraldehyde 3-phosphate d ehydrogenase (GAPDH), using laser densitometry, phosphorimaging, and l iquid scintillation counting. In this report we demonstrate that incor poration of [alpha-P-32] dCTP into the PCR amplicons followed by band excision from agarose gels, and counting by liquid-scintillation is bo th cost-effective and reproducible method for mRNA transcript semiquan titation.