Background: NNE (non-neuronal alpha-enolase) is a glycolytic enzyme detected in most tissues. NSE (neuron-specific gamma-enolase) is detected in normal neurons and tumors such as neuroblastoma. Staining with antibodies against NSE is therefore used to detect neuroblastoma cells invading bone marrow. Since staining of normal leukocytes has been reported we asked whether bona fide NSE is in fact expressed in normal blood and marrow.
Experimental design: We designed nested coding region specific primers for NSE and NNE and, after reverse transcription of mRNA, we amplified the coding region between these primers in a semi-nested polymerase chain reaction. In order to distinguish both iso-mRNAs from each other, we amplified a long (1,047 bp) template in a first round of 30 cycles with primers specific for NNE or NSE. One percent of this product was used in a second round of 30 cycles in which both sense primers and two nested anti-sense primers of alternate specificities yielding shorter products of discernible sizes (768 bp or 619 bp) were added together in the same reaction tube. With this combination of four primers, only that shorter product was amplified to visibility, the specificity of which was homologous to the template produced in the first 30 cycles. Restriction enzyme digestion of the amplified products was used to verify this polymerase chain reaction-based approach for the distinction of isoforms of RNA.
Results: This semi-nested polymerase chain reaction clearly allows for the distinction of mRNA for NNE or NSE and shows the presence of transcripts for NSE in normal human leukocytes from blood and bone marrow.
Conclusions: This method exploiting short stretches of nucleotide differences in the coding regions for priming can more generally be applied to the distinction of all isoforms of RNA where nested specific primers can be designed. However, the presence of NSE specific transcripts in normal human leukocytes invalidates the use of this highly sensitive method as a disease marker in neuroblastoma.