Comparative non-radioactive RT-PCR assay: An approach to study the neurosteroids biosynthetic pathway in humans

Polymerase chain reaction (PCR) is a powerful tool for qualitative evaluation of nucleic acid expression. PCR has been widely applied to measure DNA and RNA messages expression. Neurosteroids synthesized in the nervous system are potent modulators of synaptic activity and have been implicated in several neuropsychiatric disorders. To examine the possibility of an altered expression of the neurosteroidogenic metabolic enzymes in neurological diseases (like Parkinson's disease, PD) we developed a comparative non-radioactive RT-PCR assay to detect the mRNA levels of the peripheral benzodiazepine receptor, the 5α-reductase type 1 and 3α-hydroxysteroid-oxidoreductase type 1 and 2 in lymphocytes obtained from PD patients. The results were compared with that obtained from simultaneous quantification of progesterone, 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone in the plasma and cerebro-spinal fluid of the same individuals using a gas chromatography mass spectrometry (GC/MS) technique. We found a significant decrease of the rate-limiting enzyme 5α-R1 along with a significant decrease in plasma and CSF of the 3α,5α-tetrahydroprogesterone and of the 5α-dihydroprogesterone. Comparative RT-PCR assay, along with complimentary techniques (i.e. GC/MS), has the sensitivity, selectivity and dynamic range to allow specific and reliable quantization of the enzymes involved in the neurosteroids pathway and represent a valuable tool to assess their expression in human neuropsychiatric conditions.