Production of RNA for transcriptomic analysis from mouse spinal cord motor neuron cell bodies by laser capture microdissection.
Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.
Department of Genetics, Yale School of Medicine.
Urmi
U
Bandyopadhyay
Wayne A
WA
Fenton
Arthur L
AL
Horwich
Maria
M
Nagy
D000818:Animals; D001399:Azure Stains / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D061346:Laser Capture Microdissection / Q000379:methods*; D051379:Mice; D009474:Neurons / Q000737:chemistry / Q000166:cytology*; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013116:Spinal Cord / Q000737:chemistry / Q000166:cytology*
D001399:Azure Stains; D012313:RNA; C025819:Azure B