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Laser Capture Microscopy and real time RT‐PCR: Phenotyping spinally projecting brainstem and forebrain neurons
Author(s) -
Weissbach Sonja P.,
Foley Charles M.,
Heesch Cheryl M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a363-d
Subject(s) - rostral ventrolateral medulla , brainstem , forebrain , complementary dna , real time polymerase chain reaction , microbiology and biotechnology , biology , chemistry , medulla oblongata , medicine , anatomy , endocrinology , neuroscience , biochemistry , central nervous system , gene
These experiments demonstrate the use of laser capture microscopy (LCM) and real time RT‐PCR to evaluate neurotransmitters, neuromodulators and receptor expression in spinally projecting neurons in the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus of the hypothalamus (PVN). Fluororuby (1%, 90 nl) was microinjected into the intermediolateral cell column of the thoracic spinal cord (T1 – T2) 7 days prior to harvesting brains. Brains were frozen, 10 μ sections obtained from forebrain and brainstem, and dehydrated. Retrogradely labeled neurons were visualized and microdissected from the RVLM and PVN using Capsure MacroLCM caps and a PixCell IIe laser capture microscope (20X; Spot size = 7.5 μ). RNA was extracted and cDNA synthesized using standard procedures. Real time RT‐PCR (Cepheid Smart Cycler II) using SYBR green was performed for mRNAs of GAPDH (house keeping gene), GABA A receptor subunits and nNOS. Standards for target PCR products had been previously cloned, isolated, purified, and quantified with spectrophotometry. Serial dilutions of the appropriate standards were used to quantitate amount of product. Caps with ~15–25 cells yielded 24 μl of cDNA and quantifiable amounts of GAPDH, GABA A α 1 & δ subunit, and nNOS in 1 μl aliquots. Values are ratio to GAPDH from the same cap (pmol/nmol GAPDH). [RVLM: α 1 = 251.5; delta; = 223.7; nNOS= 34.6; PVN: α 1 = 192.8; delta; = 66.4; nNOS= 98.9]. It is predicted that 6‐10 mRNA’s can be determined from each cap of pooled cells. Thus, LCM and real time RT‐PCR will allow for detailed phenotyping of neurons involved in control of sympathetic outflow. (NIH HL36245)