TY - JOUR
T1 - Comparative non-radioactive RT-PCR assay
T2 - An approach to study the neurosteroids biosynthetic pathway in humans
AU - Luchetti, Sabina
AU - di Michele, Flavia
AU - Romeo, Elena
AU - Brusa, Livia
AU - Bernardi, Giorgio
AU - Cummings, Brian J.
AU - Longone, Patrizia
PY - 2006/6/15
Y1 - 2006/6/15
N2 - 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.
AB - 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.
KW - 3α-HSOR 1-2
KW - 5α-Reductase 1
KW - GC-MS
KW - Neurosteroids
KW - Parkinson's disease
KW - PBR
KW - RT-PCR
UR - http://www.scopus.com/inward/record.url?scp=33646265027&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33646265027&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2005.11.005
DO - 10.1016/j.jneumeth.2005.11.005
M3 - Article
C2 - 16378642
AN - SCOPUS:33646265027
SN - 0165-0270
VL - 153
SP - 290
EP - 298
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 2
ER -