RT-PCR ELISA method for the analysis of neurotrophin mRNA expression in brain and peripheral tissues.
The levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) in brain and periphery are susceptible to changes during development and as result of different physiopathological conditions, such as stress and aging and during the onset and progression of neurological and autoimmune diseases. Despite the sensitive methods for measurement of neurotrophin protein levels in different tissues, no easily applicable methods to evaluate changes in the level of NGF and BDNF mRNA expression within physiological range have been described. This study reports the development of a reproducible and simple procedure for measurement of neurotrophin mRNA expression in brain and peripheral tissues based upon an enzyme linked immunosorbent assay (ELISA) detection system of reverse transcriptase-polymerase chain reaction (RT-PCR) products. The major advantages of this RT-PCR ELISA procedure is to allow the co-amplification of diverse mRNAs starting from small amounts of tissues; to contemporaneously test a large number of samples; to be rapid and to use only commercial reagents and widely available equipment. The procedure could also be useful in studies addressed to measure the pattern of expression of molecules involved in the pathogenesis of neurodegenerative and inflammatory diseases, such as neuropeptides and cytokines.
Tirassa
P
P
Institute of Neurobiology (CNR), Viale C. Marx, 15/43, 00137, Rome, Italy.
Manni
L
L
Stenfors
C
C
Lundeberg
T
T
Aloe
L
L
D000818:Animals; D001921:Brain / Q000378:metabolism*; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis / Q000235:genetics; D002540:Cerebral Cortex / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006624:Hippocampus / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012867:Skin / Q000378:metabolism; D013154:Spleen / Q000378:metabolism; D013363:Submandibular Gland / Q000378:metabolism
D019208:Brain-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases