Production of high quality brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) RNA from isolated populations of rat spinal cord motor neurons obtained by Laser Capture Microdissection (LCM).
The mammalian central nervous system (CNS) is composed of multiple cellular elements, making it challenging to segregate one particular cell type to study their gene expression profile. For instance, as motor neurons represent only 5-10% of the total cell population of the spinal cord, meaningful transcriptional analysis on these neurons is almost impossible to achieve from homogenized spinal cord tissue. A major challenge faced by scientists is to obtain good quality RNA from small amounts of starting material. In this paper, we used Laser Capture Microdissection (LCM) techniques to identify and isolate spinal cord motor neurons. The present analysis revealed that perfusion with paraformaldehyde (PFA) does not alter RNA quality. RNA integrity numbers (RINs) of tissue samples from rubrospinal tract (RST)-transected, intact spinal cord or from whole spinal cord homogenate were all above 8, which indicates intact, high-quality RNA. Levels of mRNA for brain-derived neurotrophic factor (BDNF) or for its tropomyosin receptor kinase B (TrkB) were not affected by rubrospinal tract (RST) transection, a surgical procedure that deprive motor neurons from one of their main supraspinal input. The isolation of pure populations of neurons with LCM techniques allows for robust transcriptional characterization that cannot be achieved with spinal cord homogenates. Such preparations of pure population of motor neurons will provide valuable tools to advance our understanding of the molecular mechanisms underlying spinal cord injury and neuromuscular diseases. In the near future, LCM techniques might be instrumental to the success of gene therapy for these debilitating conditions.
Translational Neuroscience Facility, School of Medical Sciences, UNSW Australia, Sydney, New South Wales 2052, Australia.
Prachi
P
Mehta
Translational Neuroscience Facility, School of Medical Sciences, UNSW Australia, Sydney, New South Wales 2052, Australia.
Brian
B
Premkumar
Translational Neuroscience Facility, School of Medical Sciences, UNSW Australia, Sydney, New South Wales 2052, Australia. Electronic address: renee.morris@unsw.edu.au.
Renée
R
Morris
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000302:isolation & purification*; D005260:Female; D061346:Laser Capture Microdissection / Q000379:methods*; D009046:Motor Neurons / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification; D051381:Rats; D020318:Rats, Long-Evans; D020813:Receptor, trkB / Q000302:isolation & purification*; D013116:Spinal Cord / Q000378:metabolism*
D019208:Brain-Derived Neurotrophic Factor; D012333:RNA, Messenger; D012313:RNA; D020813:Receptor, trkB
Brain-derived neurotrophic factor (BDNF); Gene expression; Laser capture microdissection (LCM); Motor neurons; Rubrospinal tract (RST); Spinal cord; Tropomyosin receptor kinase B (TrkB)