Comparison of hybridization methods and real-time PCR: their value in animal cell line characterization
Porphobilinogen deaminase (PBG-D), the third enzyme in the heme synthetic pathway, possesses two isoforms encoded by distinct mRNAs that are the result of transcription of a single gene from two promoters through differential splicing. During erythroid differentiation, only the expression of the erythroid-specific isoform (E-PBGD) was increased. A system was developed to evaluate genetic expression of E-PBGD in samples limited in cell number and/or mRNA copy. Total RNA from human cells was reverse-transcribed and amplified by the polymerase chain reaction in the same tube with an internal standard that is an in vitro transcript of a cDNA differing from its sample counterpart by a few restriction sites and 24 bp (10%) in the target region. The primers spanned through regions where sample and standard templates were identical in sequence. Amplified templates were resolved by restriction enzyme digestion and gel electrophoresis and quantified by densitometer tracing of corresponding bands on autoradiograms. When an appropriate amount of internal standard is present in the reaction mixture, the ratio of amplified sample versus standard template is proportional to the amount of sample RNA and it is therefore possible to calculate the number of specific mRNA molecules in the original sample.